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This electronic thesis or dissertation has beendownloaded from Explore Bristol Research,http://research-information.bristol.ac.uk
Author:Noll, Madeleine E
Title:The Control of Stomoxys calcitrans (Stable Flies) with Essential Oils
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1
The Control of Stomoxys calcitrans (Stable Flies) with Essential Oils
Madeleine Noll
A dissertation submitted to the University of Bristol in accordance with the requirements for award of the degree of MSc (Res) in the
Faculty of Life Sciences, School of Biological Sciences.
10th August 2020
Word count: 14,764
2
Abstract
Stable flies are important hematophagous ectoparasites due to their broad range of mammalian hosts and world-wide distribution. As a result of their interrupted feeding behaviour, stable fly biting can result in a suite of direct and indirect adverse effects for their hosts. When densities are high, stable fly control is important particularly in dairy and beef cattle systems, on economic and welfare grounds. However, recently, the negative environmental and health consequences associated with exposure to conventional synthetic insecticides have become evident as well as the increasing development of resistance. Consequently, there is a need for an environmentally substantiable and effective alternative mechanism for stable fly control to be identified. The work set out in this thesis aimed to evaluate the efficacy of essential oils as insecticides and repellents for stable flies.
A semi-quantitative literature analysis of essential oils against biting flies suggested that lavender and tea tree oils were likely to be effective and hence these oils were chosen for investigation against stable flies. Using laboratory bioassays using laboratory bioassays in the stable fly, Stomoxys calcitrans, 5% (v/v) lavender and tea tree essential oils with ethanol excipient, caused 100% mortality for 4 and 6 h after exposure, respectively. In repellency bioassays, 5% (v/v) lavender and tea tree oils were able to deter 83.3% and 90% of stable flies from crossing an impregnated filter paper funnel for 1 h, respectively. The repellency of these essential oils was greater than that of a commercial repellent (20% DEET) which repelled 63.3% of flies for 1 h. The effectiveness of these oils in vitro, suggests that future work should focus on examining their potential in vivo. If effective in the field, these oils pose as viable alternatives to conventional synthetic treatments used in high value animal husbandry, particularly if issues associated with their cost and limited residual activity can be overcome.
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Dedication and acknowledgements
There are several people I would like to acknowledge for their assistance and support during the
research and writing of this project. Firstly, I would like to express my gratitude to my supervisor
Professor Richard Wall for his encouragement, support and invaluable guidance throughout this
project. I would also like to thank Dr Bryony Sands and Oliver Souter for sharing their knowledge with
me which helped tremendously. I would also like to extend my thanks to my fellow MSc students,
Katie Bryer, Amber-Rose Cooper and many others for their continual encouragement, support and for
making this such a fun year.
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Author’s Declaration
I declare that the work in this dissertation was carried out in accordance with the requirements of the
University's Regulations and Code of Practice for Research Degree Programmes and that it has not
been submitted for any other academic award. Except where indicated by specific reference in the
text, the work is the candidate's own work. Work done in collaboration with, or with the assistance
of, others, is indicated as such. Any views expressed in the dissertation are those of the author.
Signed: Madeleine Noll Date: 10th August 2020
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Table of Contents
Chapter 1: Stomoxys calcitrans: veterinary importance and control
1.1 Stomoxys calcitrans………………………………………………………………………………………………………………………8
1.2 Veterinary Importance…………………….…………………………………………………………………………………………10
1.2.1 Direct effects…..…………………………………………………………………………………….……………………10
1.2.2 Indirect effects……………………………………………………………………....…………………….……….……11
1.2.3 Economic impact………………………………………………………………………………………………………..12
1.3 Stable fly control…………………………………………………………………………………………………………………………13
1.3.1 Chemical.………………………………………………………………………………….……………..…………………13
1.3.2 Mechanical……………………………………………………………………………….…………..……………………14
1.3.3 Biological…………………………………………………………………………………….…………..…………………14
1.3.4 Cultural.…………………………………………………………………………………………….………….……………15
1.4 Botanical Pesticides……………………………………………………………………………………………………………………15
1.4.1 Essential oils……………………………………………………………………………………………………….………16
1.5 Insecticidal properties of essential oils against biting flies………………………………………………………………24
1.5.1 Evaluating the efficacy of essential oils against biting flies……………....……….……….………25
1.5.2 The use of essential oils against stable flies……………………..…….……….………………………….26
1.6 Aims………………………………………………………………………………………………………………………………………..…29
Chapter 2: Efficacy of the essential oils against Stomoxys calcitrans in in vitro experiments
2.1 Introduction….................................................................................................................................30
2.2 Materials and methods…................................................................................................................30
2.2.1 Stomoxys calcitrans…......................................................................................................30
2.2.2 Essential oils….................................................................................................................31
2.2.3 Insecticidal bioassay…………………………………………………....................................................31
2.2.4 Repellency bioassay…………………………………………………....................................................32
2.2.5 Statistical analysis ……………………………………………………………............................................34
2.3 Results…..........................................................................................................................................34
2.3.1 Essential Oils ………………………………………………………….......................................................34
2.3.2 Insecticidal bioassay…………………………………………………………............................................35
2.3.3 Repellency bioassay…………………………………………………………............................................35
2.4 Discussion….....................................................................................................................................38
Chapter 3: Discussion
3.1 General Discussion….......................................................................................................................41
3.2 Conclusions…………….......................................................................................................................45
Appendix
Appendix I. An enumeration of essential oils which have been investigated for their repellent or
insecticidal properties against biting flies of veterinary importance…………………………………………………66
6
List of Figures
Chapter 1: Introduction: Stomoxys calcitrans, their veterinary importance and control mechanisms
1. Female Stomoxys calcitrans L. (From Cumming, 1998).………………………..……………………………………..8
Chapter 2: Efficacy of the essential oils against Stomoxys calcitrans in in vitro experiments
2.1. The experimental apparatus used to determine if essential oils were a feeding deterrent to
Stomoxys calcitrans. (1) Blood soaked cotton wool placed on (2) a mesh-ended plastic pint cup which
formed the upper feeding chamber. (3) The funnel was constructed from a 2L plastic bottle neck
containing a treated filter paper. (4) A plastic pint cup connected to (5) a half-pint plastic cup with a
mesh bottom which formed the entrance chamber. (6) Electric fan for airflow through the
apparatus.……………………………………………………………………………………………………………………………………….33
2.2 Mortality (mean ±SE) of Stomoxys calcitran at 15, 30 and 45 min and 1, 2, 4, and 6 h post-exposure
to filter papers impregnated with 5% (v/v) lavender essential oil (○), 5% (v/v) tea tree essential oil (▲)
and absolute ethanol (excipient only negative control) (■). Points have been offset and joined for
clarity……………………………………………………………………………………………….………..……………………………………36
2.3 The number of Stomoxys calcitrans (mean ±SE) that reached the end chamber of an olfactometer
containing blood-soaked cotton wool after passing a filter paper funnel impregnated with 5% (v/v)
lavender essential oil (○), 5% (v/v) tea tree essential oil (▲), DEET (20% v/v) positive control (●),
absolute ethanol excipient-only negative control (□) and untreated negative control (■) at baseline, 5,
15, 30, 45 and 60 min. Points have been offset and joined for clarity……………………………………………….37
7
List of Tables
Chapter 1: Introduction: Stomoxys calcitrans, their veterinary importance and control mechanisms
1. Three proposed neurological modes of action of essential oils and their components on insect
nervous systems..………………………………….…………………………………………………………………………………………19
Chapter 1: Insecticidal and repellent effects of lavender, Lavandula angustifolia, and tea tree, Melaleuca alternifolia, essential oils against stable flies.
2. The number of points allocated to the top five performing essential oils….…………….……………………..35
8
Chapter 1
Stomoxys calcitrans - veterinary importance and control
1.1 Stomoxys calcitrans
The genus Stomoxys (Diptera: Muscidae) is comprised of eighteen species, including the stable fly
(Stomoxys calcitrans) (Zumpt, 1973; Dsouli et al., 2011). Stomoxys, meaning ‘sharp mouth’, are unique
within the Muscidae as the adults of both sexes are obligate hematophagous ectoparasites of
mammals (Foil and Hogsette, 1994). Unlike most stomoxine species, which are found exclusively in
the tropics, stable flies are cosmopolitan pests with a world-wide distribution. This, in combination
with their extensive range of mammalian hosts makes them of veterinary importance (Zumpt, 1973;
Foil and Hogsette, 1994).
Stable flies are also referred to as ‘biting house
flies’ due to their resemblance to common house fly,
Musca domestica, in size and shape, with female adults
being approximately 7mm in length and males being
slightly smaller (Foil and Hogsette, 1994; Masmeatathip
et al., 2006). However, they are distinguishable due to
their colouration; they are lighter and greyer in colour
with four longitudinal darkened stripes on their thorax
and black checkering on their abdomen (Fig. 1)
(Masmeatathip et al., 2006). Furthermore, stable flies
have a labellum which is equipped with teeth and their
proboscis is forward-facing, slender and sharp to assist in
piercing skin (Todd, 1964; Patra et al., 2018). These flies
are also sexually dimorphic, and the compound eyes of male stable flies are closer together compared
to females (Zeil, 1982). Collectively, these characteristics can assist in the identification of stable flies.
However, as with all holometabolous insects, the conditions in which larvae develop can significantly
affect their adult size and fitness (Baleba et al., 2020).
There are four life-cycle stages: eggs, larvae, pupae and adults. Following their first blood
meal, male stable flies can successfully inseminate females; this usually occurs around four days after
Figure 1. Female Stomoxys calcitrans L. (from Cumming, 1998).
9
emergence and subsequently females start laying eggs at around eight days after emergence (Killough
and McKinstry, 1965; Anderson, 1978; Morrison et al., 1982). Gravid females lay clusters (~20-100) of
white elliptical eggs within specific and individual oviposition substrates (Todd, 1964; Baleba et al.,
2020). These oviposition sites usually consist of putrefying organic materials, such as decaying grass,
silage and hay, which are inherently found in close association with livestock (Meyer and Petersen,
1983). Once hatched, the translucent larvae bury into this medium and successively moult through
three larval stages and subsequently pupariate into a reddish-brown pupation (Todd, 1964; Gilles et
al., 2005). Adults emerge 7-14 days after pupariation and can fly within one hour (Foil and Hogsette,
1994; Berry and Kunz, 1997). Notably, the duration of stable fly development is highly dependent on
environmental conditions, such as temperature and availability of recourses (Florez-Cuadros et al.,
2019). For example, at 15°C the time required from the deposition of an egg to the emergence of an
adult is 71 days, whereas it only takes 13 days at 30°C (Gilles et al., 2005). Similarly, the average
lifespan of stable flies varies, with wild flies living for approximately two weeks compared to over four
weeks for those kept in a laboratory (Killough and McKinstry, 1965; Berry and Kunz, 1997).
Over the course of their life, stable flies usually take between one and three blood meals per
day, each lasting around three minutes and imbibing 11-15 μL of the host’s blood (Schowalter and
Klowden, 1979; Harris et al., 1974). These flies feed on a wide range of warm-blooded mammals,
particularly bovids and equids (Patra et al., 2018). As a synanthropic pest, common hosts also include
domesticated animals, such as cats and dogs. They generally bite the thinner-skinned regions of their
host due to easier penetration and higher density of capillaries near the surface. In bovids and equids,
the front legs and underbelly are commonly bitten sites, whereas the tips of ears are more usually
attacked in canines and felines (Yeruham and Braverman, 1995).
Due to their dependency on blood meals, stable flies usually aggregate in locations where
hosts congregate, such as feed lots, water stations and outside shelters (Showler and Osbrink, 2015).
As ectotherms, these flies are attracted to brightly illuminated surfaces, with high reflectance of
ultraviolet light and iridescence, and rest on these surfaces during the morning to become active
during the midday sun (Buschman and Patterson, 1981; Agee and Patterson, 1983). They usually
employ a sit-and-wait predatory method, but they can make short distance (<1.6 km) flights in search
of hosts and oviposition sites (Hogsette 1983; Showler and Osbrink, 2015). It has become evident that
stable flies also perform long-distance dispersals; for example, one population of stable flies was
found to have relocated 225km from inland Florida to the coast (Hogsette and Ruff, 1985). However,
10
such long-range migrations are usually considered to be passive movements, driven by weather. The
weather can not only influence the distribution of these flies, but also their abundance (Lysk, 1993).
Stable fly abundance corresponds to several climatic factors and hence seasonal peak
abundance often varies with location, depending on local conditions (Lysk, 1993; Machtinger et al.,
2016). In South-west England their numbers increase during summer months and peak in late August,
early September (Parravani et al., 2019). Despite Parravani et al., (2019) finding no relationship
between any climatic factors and their abundance, others have found that temperature and
precipitation are strong predictors of stable fly prevalence (Lysk, 1993; Skovgård and Nachman, 2012).
In a 13-year study in Nebraska, temperature and precipitation were found to be responsible for 72%
of the variation in stable fly population, with populations peaking during the warmer season (Taylor
et al., 2017). In many agricultural environments increases in stable fly numbers during the summer
months correspond to when livestock are more likely to be turned out and thus become easier targets
for flies.
1.2 Veterinary Importance
1.2.1 Direct effects
The stable fly is one of the most problematic biting flies due to its irritability to hosts. By flying
around and landing on their hosts, stable flies induce a range of defensive host behaviours, such as
tail swishing, foot stamping, muscle twitching, head throwing, aggregating in groups and seeking
protection by moving to other, less infested areas (Mullens et al., 2017; El Ashmawy et al., 2019;
Kohari et al., 2020). However, these behaviours come at a cost to hosts. For example, a host’s energy
expenditure is increased and their foraging ability and time, thus their food and energy intake are
reduced (Dougherty et al., 1993). It has been claimed that cattle increase their bite size and herbage
intake in order to compensate for this reduction in foraging time, although there is little clear evidence
to support this (Dougherty et al., 1994). These defensive behaviours may also result in injury; the
aggregation of cattle during ‘bunching’ behaviours, whereby individuals migrate centrally into a group
to seek greater protection, can lead to increases in injury and heat stress (Wieman et al., 1992).
Avoidance behaviours may also inadvertently prolong the period of annoyance as feeding flies are
interrupted before completing their blood meal and thus require multiple feeds.
The close proximity of stable flies can also increase the physiological stress experienced by
their hosts (Colwell et al., 1997). In dairy cattle, there is a linear relationship between the cortisol
concentrations of a cow and the number of stable flies residing on the animal; cattle experiencing on
11
average 0 or 26 flies/day were shown to have cortisol concentrations of 2.5 ng/mL and 56 ng/mL,
respectively (Vitela-Mendoza et al., 2016). Furthermore, indicators of stress, including increases in
heart and respiration rates as well as rectal temperatures, have been recorded in cattle exposed to 25
flies (Schwinghammer et al., 1987). Incidentally, stable flies are attracted to volatile compounds found
in breath, such as carbon dioxide and 1-octen-3-ol, thus increases in breathing rates could potentially
increase attraction (Hieu et al., 2014). Furthermore, the painful bite of a stable fly and associated loss
of blood can further increase the physiological stress experienced by a host (Colwell et al., 1997). The
loss of blood is not limited to that imbibed by stable flies, there is also pooling of blood around the
bite site due to stable fly probing. Together, elevated stress levels and amplified body movements,
increase the energy expenditure of hosts and thus reduces their available reserves for growth,
maintenance and reproduction.
During the physical act of feeding, stable flies excrete saliva which contains pharmacologically
active molecules that inhibit blood clotting and increase vasodilation (Swist et al., 2002). Components
of this saliva may also initiate immunological responses, which can lead to immunosuppression and
allergic reactions in susceptible animals. One study demonstrated that an intradermal injection of 2.4
mg of stable fly protein can cause immediate hypersensitivity in susceptible horses, and thus could
play a role in the aetiology of sweet itch (Braverman et al., 1983).
1.2.2 Indirect effects
Stable fly feeding may also have a number of indirect effects on their hosts. The wounds
created by stable flies can become infected, as evidenced by the necrotising dermatitis on the tips of
dog ears and exudative dermatitis on horses and cattle lower legs (Yeruham and Braverman, 1995;
Urban and Broce, 1998). These lesions and cutaneous infections can secondarily aid the recruitment
of other hematophagous parasites and increase the incidence of infections, such as myasis (Yeruham
and Braverman, 1995).
Furthermore, stable flies may indirectly affect their host due to their role in the epidemiology
of pathogens. The transmission of pathogens could be facilitated by the interrupted feeding habits of
stable flies as they regurgitate the blood of their previous host at their new feeding site (Butler et al.,
1977). However, despite numerous studies, there is relatively little good data to support this vectoral
role, despite many claims to the contrary. For example, Turell et al. (2010) concluded stable flies could
act as mechanical vectors of Rift Valley fever virus as they are capable of transmitting the virus from
highly infected to susceptible hamsters under laboratory conditions. However, these results have not
12
been confirmed in field populations. Similarly, the discovery of Trypanosoma DNA in stable flies in
Nigeria is not definite evidence that they are mechanical vectors (Odeniran et al., 2019) since the
presence of pathogen DNA only in the blood meal shows only its presence not whether it can be
transmitted. More convincingly, capripox viruses were shown to survive within stable flies for six days
after ingestion and be transmitted to susceptible goats and sheep under experimental conditions
(Mellor et al., 1987). However, at present, there is no epidemiological evidence for the mechanical
vectoral capabilities of stable flies in nature.
Female stable flies have also been reported as intermediate hosts of the nematode,
Habronema microstoma (Traversa et al., 2008). Despite including field experiments, this conclusion
comes primarily from positive polymerase chain reaction samples, rather than definitive evidence of
transmission of the nematode. To fully elucidate the potential veterinary importance of these flies,
their role in the epidemiology of transmittable pathogens warrants further, more detailed
investigation.
1.2.3 Economic impact
The effects of stable flies on their hosts have not only great veterinary importance but are
also economically significant due to losses in yield. These economic consequences have been most
extensively studied in cattle due to their commercial importance. Unfortunately, studies designed to
quantify these effects are often inconclusive often because they use inappropriate controls (Shaw and
Atkeson, 1943; Campbell et al., 2001), unsuitable environmental chambers (Miller et al., 1973) and
include additional biting fly species in their analysis (Cutkomp and Harvey, 1958; Morgan and Bailie,
1980) and are thus not suitable for evaluation. Taylor and colleagues (2012) collated the results from
reliable studies and estimated that as few as 10 flies/cow/day can cause significant economic losses.
In US dairies with a high stable fly abundance, it was estimated that losses of 139kg milk/cow/year
can be expected, which equates to $40 per animal (Taylor et al., 2012). In the meat industry, individual
cattle under stable fly attack can incur annual weight losses of 26kg, equalling $48 (Taylor et al., 2012).
In total, stable flies cost the United States of America cattle industry $2.2 billion annually (Taylor et
al., 2012). These estimates are based on US agricultural prices in 2009, and only relate to cattle and
hence are difficult to extrapolate to other systems. Thus, more research on the economic effects of
stable flies on a wider range of host animals would be of value.
1.3 Stable Fly Control
1.3.1 Chemical
13
Since their development and introduction, synthetic chemicals have become strongly
integrated into nuisance fly control programmes for livestock. Feed-additive insecticides, such as the
organophosphate tetrachlorvinphos, are widely used as they prevent larval development in manure.
However, these treatments are often ineffective against stable flies because of the variety of
oviposition sites that this species uses (Campbell, 1977). Alternatively, surfaces surrounding livestock
can be treated directly with environmental insecticide preparations with the aim of discouraging
oviposition and increasing mortality (Hogsette et al., 1987). However, due to the transient nature of
their oviposition media and tendency of larvae to bury, this method of application is often impractical,
inefficient and environmentally damaging. At present, the most effective mechanisms for stable fly
control are pour-on formulations, including organophosphates, permethrins and pyrethroids
(Muraleedharan, 2005; Mottet et al., 2018). However, due to short residual activities, most pour-on
treatments require repeated application which is expensive and, therefore, is only justified during
periods of high stable fly abundance (Foil and Hogsette, 1994).
The repeated application of synthetic chemicals can cause both environmental and health
hazards, including non-target effects and the contamination of livestock products (milk and meat)
(Gebremichael et al., 2013; Pouokam et al., 2017; Sands et al., 2018). For example, synthetic
pyrethroids can cause non-target effects on both terrestrial and aquatic organisms, including
biologically important species, such as the dung beetle (Uddin et al., 2016; Sands et al., 2018). In
addition to mortality, dung beetles exposed to these chemicals can show an array of sublethal effects,
including reduced motility and impaired reproductive output (Sands et al., 2018; Weaving et al., 2020).
The ecosystem services provided by dung beetles was estimated to save the cattle industry, in the
United Kingdom, £367 million annually (Beynon et al., 2015). As with the dung beetle, other important
species are adversely affected by the routine application of insecticides and consequently there have
been efforts to minimise the use of synthetic pesticides.
A range of alternative application methods have been developed which may have less
environmental impact. For example, insecticide-impregnated ear tags are commonly used against flies
on cattle (Hogsette and Ruff, 1986). However, as these devices rely on the self-grooming and
movements of cattle for application, the neck and shoulders receive the greatest coverage and hence
these devices are inadequate against leg biting stable flies (Beadleas et al., 1977). The use of
permethrin impregnated tail tags was investigated on dairy cattle and it was concluded that this
mechanism was much more effective, eliminating stable flies within 24 hours (Hogsette et al., 1987).
14
Despite this success, these tail tags have not been marketed due to their short residual activity and
increasing incidence of insecticide resistance in flies.
The progressive development of pesticide resistance in stable flies means these routinely
administered synthetic treatments are becoming ever more ineffectual in many parts of the world
(Cilek and Greene, 1994; Pitzer et al., 2010). Salem and colleagues (2012) investigated the level of
resistance in stable flies collected from an organic and conventionally treated farm to six chemical
treatments: cypermethrin, fenvalerate, permethrin, λ-cyhalothrin, deltamethrin and phoxim. Flies
from the conventional farm were resistant to the five synthetic pyrethroids and the authors suggested
using alternative organophosphate treatments (Salem et al., 2012). However, the application of
additional insecticides will increase resistance and enhance the potential for environmental damage
caused by their application. Hence there is a need for new pest management approaches which are
both effective and sustainable.
1.3.2 Mechanical
There has been considerable interest in the mechanical control of stable flies through
pesticide-free traps (Taylor and Berkebile, 2006). The majority of these traps exploit the flies’ optical
attraction to polarised sunlight by coating reflective materials with an adhesive layer (Williams, 1973;
Taylor and Berkebile, 2006; Turell et al., 2010; Hogsette and Kline, 2017). Additional attractants, such
as carbon dioxide, have been evaluated as complementary olfactory stimuli, but have been considered
unnecessary as the equipment required for their production outweighs the additional gain in the
number of flies caught (Cilek, 1999). The effectiveness of these traps has been demonstrated under
a wide range of conditions. However, to manage a stable fly population effectively large numbers must
be caught in close proximity to hosts and achieving this in large-scale agricultural settings is difficult
(Ose and Hogsette, 2014; Hogsette and Kline, 2017; Hogsette and Ose, 2017). Hence, it is generally
concluded that, while traps are effective for monitoring and supressing populations, they need to be
used in conjunction with other techniques for population elimination (Hogsette and Kline, 2017).
1.3.3 Biological
Parasitoids, both naturally occurring and introduced, have been considered as alternative
biological control strategies against stable fly infestations. However, their effectiveness varies greatly.
On organic dairy farms in Denmark, a bi-weekly release of Spalangia cameroni distinctly reduced the
number of stable flies per animal (Skovgård, 2004). However, when sentinel pupae of Muscidifurax
raptor and S. nigroaenea were introduced into outdoor feedlots in Nebraska, even at fivefold the
15
recommended rate, there was no reduction in the stable fly population (Andress and Campbell, 1994).
This variation can be explained by differences in climatic conditions and animal husbandry practices.
However, a three-year study in Illinois showed between year variation in the effectiveness of S.
nigroaenea and M. raptor at the same location, suggesting limited and inconsistent efficacy (Weinzierl
and Jones, 1998). The level of parasitism provided by these parasitoids positively correlates with
temperature, thus affecting their effectiveness across a season (Skovgård, 2004). Interestingly,
individual parasitoid species are locally adapted to attacking stable fly larvae in different substrates
and conditions, thus for optimum success, enhancing naturally occurring populations may be the most
effective mechanism (Pitzer et al., 2011). Therefore, for the appropriate use of parasitoids, there must
be individual assessments and continued monitoring of effectiveness, which may be impractical and
expensive.
1.3.4 Cultural
One of the most effective approaches to stable fly control involves adopting higher sanitation
standards in agricultural areas (Hogsette et al., 1987). Repelling and killing adult stable flies only
causes periodic suppressions in the population, since developing eggs and larvae will subsequently
emerge. Therefore, limiting the availability of oviposition media is an effective approach to a
successful long-term pest management. This can be achieved by stacking the hay in dry places to
ensure humidity is too low for stable fly eggs (Hogsette et al., 1987). Furthermore, improving water
and manure drainage systems can reduce the larval abundance in putrefying organic materials.
Limiting the availability of oviposition media is one of the most important approaches to their control
and should be used in conjunction with other mechanisms to prevent immigration from neighbouring
sites.
1.4 Botanical Pesticides
Botanical-based pesticides have been utilised in agriculture for centuries (Isman, 2006).
Before the advent and introduction of modern chemical pesticides, traditional methods of controlling
and managing livestock ectoparasites were developed and many of these are still being used among
indigenous communities around the world (Wanzala et al., 2012). With the evident shortcomings
associated with synthetic neurotoxic pesticides, the investigation of botanical alternatives warrants
further investigation.
Considerable research interest has been focussed on the use of neem, rotenone, pyrethrum
and essential oils (Isman, 2006; Isman and Grieneisen, 2014). Neem oil and seeds from Indian neem
16
tree, Azadirachta indica, are both of great interest due to their insecticidal activity. Neem oil has a
physical mode of action which works synergistically with the oil’s disulphides to achieve a lethal effect.
However, neem seeds can function as an antifeedant as well as a moulting inhibitor (Isman, 2006).
Rotenone is produced in the rhizomes and roots of tropical legumes and prevents energy production
by acting as a mitochondrial poison (Hollingworth et al., 1993). Pyrethrum is the insecticidal oleoresin
extracted from the flowers of the Dalmatian chrysanthemum daisy, Tanacetum cinerariaefolium,
which causes the rapid knockdown of insects due to its high pyrethrin concentrations (Corcos et al.,
2019). Finally, essential oils are volatile liquids which are a plant’s natural defence mechanism against
fungi, bacteria, insects and other herbivorous pests (Isman, 2006). The potential for essential oils to
be used as a successful ectoparasite control agent will be discussed further.
1.4.1 Essential oils
Essential oils are volatile hydrophobic liquids made from a blend of 20-80 secondary
metabolites of low molecular weight which are typically extracted from aromatic plants by steam
distillation (Bakkali et al., 2008). These oils are produced, stored and secreted by highly specialized
tissues within vascular plants, such as glandular trichomes, which are specialised hair cells found on
the leaves, stem and occasionally petals of aromatic plants (Markus and Turner, 2013). The secreted
essential oils are usually characterised by high concentrations (20-70%) of two or three major
terpenoid or terpene compounds as well as trace amounts of other aliphatic and aromatic
components (Bakkali et al., 2008). Gas chromatography–mass spectrometry (GC-MS) is frequently
used to investigate the composition of these oils (Schmidt et al., 2009; Najafian, 2016). This technique
can be utilised as the gas chromatography separates molecules, which are then identified by mass
spectrometry. Fortunately, the increasing cost effectiveness of this technique has allowed most
studies investigating essential oils perform their own GC-MS prior to investigation (Isman, 2017). For
example, Schmidt et al., (2009), using GC-MS, established that the essential oil extracted from
peppermint, Mentha piperita, contained over 40 compounds, with menthol and menthone comprising
over 60% of the oil. Similarly, Nchu et al. (2012) established that Kenyan mint marigold, Tagetes
minuta, contains high proportions of monoterpenes, with cis-ocimene and beta-ocimene being major
components. Understanding the composition of essential oils is important as their pharmacological
properties have often been attributed to the blend, particularly of their major components.
The biological activity of essential oils is broad ranging, including insecticidal (Kosgei et al.,
2014), growth inhibitory (Nchu et al., 2012), antifeedant (Rajkumar et al., 2019), repellent (Mkolo and
Magano, 2007) and oviposition deterrent properties (Callander and James, 2012). The insecticidal
17
effects of essential oils have been most extensively studied on ectoparasites of medical and veterinary
importance, including mosquitos, ticks, lice, mites and flies (Ellse and Wall, 2014; Benelli and Pavela,
2018a). Notably, these insecticidal effects have been documented across multiple life cycle stages. For
example, exposure to 5% (v/v) lavender essential oil in N-lauroylsarcosine sodium salt, caused 100%
mortality in adult and nymph chewing lice, Bovicola ocellatus, and inhibited all eggs from hatching
(Sands et al., 2016). The fact that essential oils have ovicidal, larvicidal and adulticidal effects may
mean that fewer treatments could be required to eliminate target pest or parasite infestations.
Due to their volatility, essential oils can also act as deterrents or repellents (Ellse and Wall,
2014). For example, the essential oils from camomile, Matricaria chamomilla, camphor, Cinnamomum
camphora, peppermint and onion, Allium cepa, were found to repel flies from water buffalo, Bubalus
bubalis, for up to six days (Khater et al., 2009). In addition, these oils can inhibit the natural behaviours
of insects. Kenyan mint marigold essential oil (0.1 mg of neat oil) was shown to be able to deter 80.1%
of brown ear ticks, Rhipicephalus appendiculatus, from their natural questing behaviour in in vitro tick
climbing bioassays (Wanzala et al., 2014). Similarly, gravid Lucilia cuprina delayed oviposition for 6
weeks when the only available media was wool treated with 5% (v/v) tea tree, Melaleuca alternifolia,
oil (Callander and James, 2012). Lower concentrations of essential oils appear to be required to
achieve a repellent effect compared to mortality (Wanzala et al., 2012; Moyo and Masika, 2013). In
combination, the insecticidal and repellent properties of essential oils make them comparable to
several conventional control strategies.
As discussed, the efficacy of essential oils is often attributed to their major components;
however, it has been suggested that minor constituents may also have important additive and
synergistic effects (de Oliveira et al., 2017). For example, after evaluating the repellent effectiveness
of spindle pod, Cleome monophyla, oil against brown ear ticks, the authors concluded that all the
components, including minor constituents, were required to achieve the greatest efficacy (Ndungu et
al., 1995). Trace elements in essential oils may also play an important role. For example, nerolidol
(0.1%), had a repellent effect against brown ear ticks which was greater than all the major constituents
of cat’s whiskers, Gynandropsis gynandra, and the commercially available synthetic repellent, N,N-
diethyl-toluamide (DEET) (Lwande et al., 1999). Combinations of different essential oils and their
compounds can also enhance the overall biological activity (Hieu et al., 2010b). Thus, understanding
the composition of an essential oil is fundamental in understanding its biological activity.
18
While most of the literature focuses on the composition and efficacy of essential oils, less is
known about their mode of action. There is evidence that these oils can have both a contact and
fumigant insecticidal effect. The hydrophobic nature of the oils means they are able to interfere with
arthropod cuticular waxes and block spiracles which results in water stress and prevents gas exchange
(Burgess, 2009; Ellse and Wall, 2014). This effect has also been seen in lice exposed to non-essential
oils such as silicon (Talbert and Wall, 2012). However, unlike non-essential oils, exposure to essential
oil vapour can also result in mortality, implying a simultaneous neurotoxic fumigant effect (Nchu et
al., 2012; Zhu et al., 2012). There is evidence that essential oils can interfere with the central nervous
system of insects, resulting in symptoms similar to those caused by synthetic insecticides, such as
hyperextension of the appendages, paralysis and death (Table 1). These adverse outcomes can occur
if essential oils are ingested or if they pass through the insect’s spiracles or penetrate their cuticle (Zhu
et al., 2011; Callander and James, 2012).
These neurotoxic effects have mainly been attributed to three modes of action (Table 1)
(Yeom et al., 2015). The most extensively investigated method is the inhibition of acetylcholinesterase,
the enzyme responsible for hydrolysing the neurotransmitter acetylcholine. Numerous essential oil
components can competitively and non-competitively inhibit the acetylcholinesterase enzyme, in a
dose-dependent manner, and subsequently cause the deregulation of nerve impulses (Table 1).
However, it is unlikely that this is the primary route by which essential oils cause a neurotoxic effect
because at low essential oil concentrations, where neurotoxic effects of essential oils have been
recorded, there is often limited inhibition of acetylcholinesterase, and this inhibition is reversed
quickly (López and Pascual-Villalobos, 2010; Anderson and Coats, 2012).
An alternative mode of action is the allosteric modulation of the gamma-amminobutyric acid
(GABA)-gated chloride channels found in the post-synaptic neuron (Table 1) (Tong and Coats, 2010).
The binding of particular essential oil components can initiate these chloride channels to open, thus
facilitating unregulated neural impulses (Tong and Coats, 2010). However, the most convincing mode
of action, due to its prolonged efficacy, comes from the ability of essential oil components to interfere
with octopamine and tyramine (precursor to octopamine) receptors (Table 1) (Jankowska et al., 2018).
Octopamine is a multifunctional molecule in insects which has several biological roles, including a
neurohormone, neurotransmitter and a neuromodulator (Orchard, 1982). Essential oil components
are mainly agonists of octopamine receptors which initiate a cascade of effects, including increasing
intracellular cAMP and calcium levels as well as protein phosphorylation and cause the dysregulation
of the insect’s nervous system (Enan, 2001).
19
Table 1. Three proposed neurological modes of action of essential oils and their components on insect
nervous systems.
Mode of Action Mechanism Evidence of essential oils or components
Cholinergic system
Inhibition of acetylcholinesterase
1,8-cineole and terpinen-4-ol from tea tree (Melaleuca alternifolia) (Mills et al., 2004). Camphor, E-anethole, fenchone, geraniol, (−)-linalool, S-carvone, γ-terpinene (López and Pascual-Villalobos, 2010). Carvacrol and nootkatone from Alaskan yellow cedar tree (Cupressus nootkatensis) (Anderson and Coats, 2012). Oriental sweetgum (Liquidambar orientalis) and valerian (Valeriana wallichii) (Park, 2014). Artemisia ketone, β- caryophyllene, β-phellandrene, camphene, camphor, cis-ocimene and estragole (Yeom et al., 2015). Perilla aldehyde from peppermint (Mentha piperita) (Park et al., 2016).
Gamma-amminobutyric acid system
Modulation of GABA receptors
Thymol from thyme (Thymus vulgaris) (Priestley et al., 2003). Lemongrass (Cymbopogon citratus) (Costa et al., 2011). Carvacrol, pulegone, and thymol (Tong and Coats, 2010).
Octopaminergic system
Agonists and antagonists of octopaminergic receptors
Eugenol and α-terpineol (Enan, 2001). Eugenol, cinnamic alcohol, and trans-anethole (Enan, 2005).
As different components in the essential oil can act simultaneously, the neurotoxic modes of
action of these oils are not mutually exclusive. Encouragingly, the different compounds can work
additively to increase the overall biological activity of an oil. Furthermore, the use of different cellular
targets by different compounds could also slow down or even prevent the selection for resistance in
pests. Firstly, individual compounds have different modes of action even when targeting the same
20
neurotoxic pathway (Jankowska et al., 2018). For example, two monoterpenoid components found in
tea tree, carvone and fenchone, target the acetylcholinesterase enzyme at different binding sites, thus
even if an insect developed resistance against one compound, the other could still initiate a neurotoxic
effect (López et al., 2015). What is more, essential oil components target multiple neurotoxic
pathways, with multiple modes of action within each pathway, and hence the likelihood of an insect
developing resistance against all of these combinations is very unlikely. Due to their alternative modes
of action, essential oils have been shown to be effective against organophosphate-resistant strains of
tick (Costa-Júnior et al., 2016). These qualities make essential oils desirable alternatives to single
compound synthetic pesticides.
In terms of the repellent effect, essential oil components may interact with the insect’s
olfactory system and either cause adverse reactions or disrupt normal function. Usually, a volatile
odorant interacts with an olfactory receptor and co-receptor and consequently an action potential is
initiated in the olfactory receptor neuron, and this relays the information to the antennal lobe
(Andersson et al., 2015). However, compounds found in essential oils may be allosteric agonists or
antagonists of these receptors and hence could modulate the odorant receptor activity and disrupt
the ability of the insect to detect scents. Bohbot and Dickens (2010) showed that the widely used
insecticides, 2-undecanone, picaridin, DEET and ethyl butylacetylaminopropionate, inhibited specific
olfactory receptors; DEET strongly inhibited Aedes aegypti AaOR8 receptor but caused no effect on
AaOR2. The mechanism of repellence is a controversial topic as it is not fully understood, and thus
future work should continue to elucidate the mechanisms involved. In the field, essential oils may also
mask the hosts odour and hence disrupt the host-seeking behaviour of pests (Adenubi, et al., 2018).
Despite their advantages, there are limitations to the application of essential oils as botanical
pesticides. Isman (1997) claimed that the sustainable cultivation of plant material for essential oils is
a barrier to their commercialisation. One of the main issues is the requirement of large quantities of
plant material for small oil yields (0.5-6.8%) and hence the cultivation of large monocultures
(Zheljazkov et al., 2013). However, over the last two decades, there has been pioneering work in
metabolic engineering to improve essential oil yields and hence reduced the need for so much plant
material (Mahmoud and Croteau 2001; Lange et al., 2011; Wang et al., 2016). Using monoterpene
rich spike lavender, Lavandula latifolia, Muñoz-Bertomeu and colleagues (2006) overexpressed a gene
encoding the 1-deoxy-D-xylulose-5-phosphate synthase (DXS) protein, which catalyses the first steps
in the methylerythritol phosphate pathway which is the source of isopentenyl diphosphate, a terpene
precursor. The upregulation of DXS resulted in increases in essential oil yield from leaves and flowers
21
by 359% and 74.1%, respectively, compared to wild type controls. Therefore, genetic engineering
could assist in the biosynthesis of essential oils and increase yields which could in turn reduce the
need for large monocultures. It should be considered however, that essential oils are often sold as
natural alternatives, thus genetically modification may reduce their public perception and utilisation
in organic farming practices.
Furthermore, the composition, quantity and quality of essential oils can vary considerably
depending on the plant species, age, organ and vegetative cycle stage (Silvestre et al., 1997; Perry et
al., 1999) as well as the climatic and soil conditions in which the plant has been grown and harvested
(Holm et al., 1997; Masotti et al., 2003; Angioni et al., 2006; Bakkali et al., 2008). An assessment of 16
Lippia kituiensis samples, all from South Africa, revealed 5 different chemotypes: carvone, ipsenone,
linalool, myrcenone, and piperitenone rich types (Viljoen et al., 2005). Since efficacy is believed to be
attributed to its composition, variation in chemotypes poses as an inherent problem in the ability to
definitively attribute pesticidal or repellent properties to a particular plant species (Nchu et al., 2012;
Wanzala et al., 2014). Hence, to ensure homogenous essential oil compositions, all variables must be
controlled, and their composition must be assessed using gas chromatography and mass spectrometry
(GC/MS). Characterisation of essential oils not only helps identify differences within species, but also,
if an oil shows potential, other oils with analogous compositions can be investigated. At present, the
International Organization for Standardization only standardises the essential of Australian tea tree,
Melaleuca alternifolia, under the name Melaleuca terpinen-4-ol type (IOS, 2017). For the commercial
production and utilisation of essential oils as botanical pesticides further standardisation regulations
must be implemented.
The composition of an essential oil is also governed by the conditions in which it is extracted,
stored and applied (Périno-Issartier et al., 2013; Rowshan et al., 2013). Due to their high proportions
of terpenoids, essential oils are volatile substances which are highly susceptible to biodegradation
(Turek and Stintzing, 2013). This degradation is associated with the interactions between compounds,
primarily through autoxidation, which is enhanced by visible and ultraviolet light as well as
temperatures which are too high or low (Misharina et al., 2003; Misharina and Samusenko, 2008;
Najafian, 2016). For example, lavender, Lavandula officinalis, oil kept at 25 °C for four months had
significant changes to its chemical profile due to reductions in compounds of low molecular weight,
including α-pinene, β-pinene, camphene and sabinene (Najafian, 2016). In comparison, lavender oil
kept at 4 °C maintained its original chemical composition. In addition to environmental conditions,
exposure to heavy metals can accelerate the rate of autooxidation and contribute to changes in oil
22
composition, thus these oils must be kept in inert plastic containers in at cold temperatures (Turek
and Stintzing, 2013). Fortunately, unlike conventional treatments whereby sub-optimum levels can
decrease the effectiveness of the solution and lead to the increased development of resistance, this
is unlikely to occur for essential oils. Even if the composition of the essential oil has been altered, there
are still numerous compounds, using several different modes of actions which could still be effective
against the target species and the insect is still unlikely acquire resistance to these However, the
resultant changes in an oil’s composition can alter its efficacy and allergenic properties, which may be
of particular significance if they are to be applied topically to animals as pesticides (Hagvall et al., 2008;
Pavela and Sedlák, 2018). Therefore, the lack of consistency and stability in essential oil composition
is their principal limiting factor for commercial production.
The instability and volatility of essential oils also limits their environmental persistence and
residual activity and hence repeated treatments may be required to deter persistent parasite
challenges (Klauck et al., 2014; Lachance and Grange, 2014). Attention has been focused on enhancing
both the stability and residual activity of essential oils through the use of different excipients and
mechanisms such as encapsulation (Maes et al., 2019). This involves isolating biologically active
molecules from external environmental conditions by coating them in a matrix wall (Zhu et al., 2012).
This matrix wall can be composed of natural, semi-synthetic or synthetic materials, but to align with
the principles of botanical pesticides, natural coatings are preferred. Encouragingly, the encapsulation
of peppermint oil in biodegradable chitosan nanoparticles increased its thermostability compared to
pure forms by over two-fold (Shetta et al., 2019). Furthermore, encapsulation of Siparuna guianensis
with chitosan nanoparticles enhanced the duration of its larvicidal activity against yellow fever
mosquitoes, Aedes aegypti, by slowing the release of biologically active compounds (Ferreira et al.,
2019). Unfortunately, encapsulated essential oils against livestock biting flies have only been assessed
in vitro or in the field for short periods of time, thus it is unclear whether their longevity can be
improved (Zhu et al., 2010, 2014; Galli et al., 2018).
Alternatively, the addition of specific excipients can increase the stability and residual activity
of essential oils. Due to the hydrophobic nature of these oils, they are usually combined with water
and an emulsifier to ensure a homogenised solution that can be easily applied to animals (Ellse and
Wall, 2014). However, natural fixatives, such as liquid paraffin, salicylic acids and vanillin, have also
been examined as potential excipients (Tawatsin et al., 2001; Oyedele et al., 2002; Blackwell et al.,
2003). For example, when turmeric, Curcuma longa, essential oil was combined with 5% vanillin
excipients the period of time in which it provided protection from A. aegypti, Anopheles dirus and
23
Culex quinquefasciatus significantly increased (Tawatsin et al., 2001). However, Kim et al. (2012)
showed that the inclusion of vanillin with lemongrass essential oil caused notable decreases in the
electroantennogram responses of A. aegypti and associated this with the fixative overly limiting
volatilisation. Similarly, the efficacy of lavender and tea tree oils was reduced with coconut oil
excipients (Sands et al., 2016). Therefore, there is a balance between reducing the volatility and hence
increasing the residual activity of the oil while maintaining its efficacy.
From an ecotoxicological perspective the volatility of essential oils may be beneficial as it
could limit environmental contamination and bioaccumulation. The toxicity and persistence of these
botanical pesticides are less than broad spectrum synthetic pesticides and hence could cause fewer
residual effects (Muraleedharan, 2005; Khater et al., 2009). Additionally, despite exploiting insect
neurological pathways, there is interspecific variation in the effectiveness of essential oils which could
lessen their non-target effects (Campbell, 1985; López and Pascual-Villalobos, 2010). For example, S.
guianensis essential oil is an effective pesticide against green peach aphids, Myzus persicae, but has
no adverse effect on the ladybirds, Coleomegilla maculata or Eriopis connexa, their natural enemies
(Toledo et al., 2019). Furthermore, essential oils are often considered safe to fish and mammals due
to their insect specific pathways (Pavela, 2014; Pavela and Govindarajan, 2016). However, this is an
area of contention, as the majority of studies document the efficacy of one essential oil against one
insect pest and fail to incorporate other target and non-target species (Isman and Grieneisen, 2014).
Therefore, future work should prioritise investigation of the environmental and non-target effects of
these botanical pesticides.
In addition to environmental safety, understanding the vertebrate toxicity of essential oils is
of importance if they are to be administered to livestock. The majority of essential oils are considered
safe for use in humans, as shown by their widespread commercial use at low doses in food
preparation, aromatherapy and cosmetics (Turek and Stintzing, 2013). However, specific components
of oils can cause adverse reactions; for example, monoterpene ketones, such as cineole, camphor,
pulegone and thujone are powerful convulsants (Burkhard et al., 1999; Mossa et al., 2018). As
essential oil composition is so variable, even within chemotypes, all oils must be subject to individual
characterisation and toxicological profiling. For instance, East Mediterranean sage, Salvia libanotica,
harvested in the winter and spring contained different proportions of camphor and a,b-thujone, thus
could exhibit different toxicities if topically applied to an animal (Farhat et al., 2001). Furthermore,
prolonged storage and the autooxidation of essential oils can increase their toxicity and skin-
irritability, thus oils should be maintained in appropriate conditions and used before their use-by-date
24
(Hagvall et al., 2008; Pavela and Sedlák, 2018). Therefore, extensive characterisation and a
toxicological profiling of individual oils is essential prior to use on animals.
Even essential oils which have been regarded as safe have exhibited toxic effects when
topically applied to animals, particularly at high concentration. A limitation to toxicological profiling
is that the majority of studies are performed on cells in vitro or laboratory animals, such as mice and
rabbits (Zhu et al., 2009; Fouche et al., 2017, 2019). These provide an insight into the toxicity of the
oil, but in particular circumstances, these oils may react differently. For example, despite tea tree oil
being advocated as safe for use in various cosmetic and medical treatments there have been adverse
reactions to its administration, especially when at high concentrations (Yadav et al., 2017). The
application of 15 mL of 100% tea tree essential oil on the wing of a cockatiel, Nymphicus hollandicus,
caused toxicosis, convulsions, vomiting, and resulted in a coma (Vetere et al., 2020). Similar effects
were documented in three Angora cats which had each been administered 60 mL of 100% tea tree
(Bischoff and Guale, 1998). However, for uses as pesticides, essential oils are effective at low doses
(<5%), thus it is unlikely adverse reactions would occur. Nonetheless, it is important to consider the
safety of individual essential oils if they are to be applied to animals.
1.5. Insecticidal properties of essential oils against biting flies
Over the past three decades there has been extensive research into the insecticidal properties
of essential oils. Within this literature, there has been a been a preponderance of studies of the vectors
of human disease; hence mosquitoes have received a disproportionate amount of attention (Nerio et
al., 2010; Benelli and Pavela, 2018a). Within veterinary parasitology, ticks have been the most
commonly studied ectoparasite, specifically species belonging to the genera Rhipicephalus and Ixodes
(Benelli and Pavela, 2018a). Comparatively, biting flies have received limited attention (see Appendix
I). In a literature search using Scopus, Benelli and Pavela (2018a) reported that 72% of research papers
investigating the effectiveness of essential oils on biting arthropods were on mosquitos, 16% on ticks
and only 2% on biting fly species from the families Ceratopogonidea, Simulidae, Tabanidae, Muscidae,
Psychodidae and Glossinidea. Given the veterinary and economic importance of biting flies, more
research should focus on understanding their susceptibility to essential oils.
1.5.1 Evaluating the efficacy of essential oils against biting flies
The methods used for assessing essential oil efficacy against flies are extremely variable. For
the study of ticks, the Food and Agriculture Organisation (FAO) promotes the use of several well
establish techniques, such as immersion and tick climbing bioassays (FAO, 2004). However, such
25
standardised techniques do not exist for flies and the World Association for the Advancement of
Veterinary Parasitology (WAAVP) provides no specific guidelines for evaluating the efficiency of
repellents against flies (Holdsworth et al., 2006). As a consequence, comparing the efficacy of essential
oil between studies is particularly challenging.
In immersion tests, insects are submerged in an essential oil treatment, usually for one to five
min, and their consequent mortality is quantified, as well as the fecundity of treated females and egg
hatchability (FAO, 2004; Callander and James, 2012). Similarly, aliquots of essential oils can be topically
applied to flies (Zhu et al., 2011). However, in the field it is unlikely a stable fly, at any lifecycle stage,
would be directly treated in this way due to the mobility of adults and tendency for larvae bury into
their developmental media. Therefore, assessing the toxicological consequences of contact with an
essential oil treated surface is more appropriate.
The World Health Organisation (WHO) promotes the use of specific exposure kits to assess
mosquito insecticide susceptibility, which can be adapted for flies (WHO, 2018). This is a standardised
protocol which involves placing insects in tubes lined with impregnated filter papers and efficacy of
the insecticide is taken as a measure of insect mortality at intermittent time intervals. However, there
is a limited range of pre-impregnated filter papers available and the procurement of expensive specific
equipment makes this technique inaccessible to many (Aïzoun et al., 2013). There have however been
several modifications to this experimental design, including placing flies in Petri dishes containing
treated filter papers (Farnsworth et al., 1997; Cossetin et al., 2018). Alternatively, the Centre for
Disease Control (CDC) endorses the use of bottle bioassays, in which the interior of a glass bottle is
coated with a fine layer of the test compound and flies are subsequently introduced and their
mortality recorded (CDC, 2011). Lastly, due to the volatility of essential oils, their toxicity can be
assessed independently by exposing target species to the vapour of oils without contact. For example,
flies can be held in a small cage within a container which contains a treated filter paper (Zhu et al.,
2011).
Due to the mobility of flies, many studies have focussed on assessing the repellent properties
of essential oils. One method of assessment is through dual choice experiments which involve
simultaneously presenting flies with treated or untreated options and recording their movement and
behaviour; these options may include food sources or oviposition sites (Callander and James, 2012;
Baldacchino et al., 2013). However, due to the volatility of essential oils, their vapour is likely to
26
influence the behaviour flies in close proximity, and thus where the two options are presented
simultaneously even the untreated option is may be affected by the presence of the essential oil.
A better approach for determining the ability of an essential oil to deter flies is to use no-
choice experiments (Callander and James, 2012). These include skin bioassays, where test
formulations are applied to the arms of volunteers and the time taken for starved flies to feed is
recorded as the protection time (Hieu et al., 2010b). This can also include treating a blood meal or
oviposition site and recording the resultant behaviour of flies (Callander and James, 2012). However,
there is considerable variation in the experimental design adopted in different studies, which can
make comparison between experiments difficult. For example, variation in extraction techniques,
excipients, assays, concentrations and time periods makes the replicability and evaluation of research
in this area challenging (Ellse and Wall, 2014). The standardisation of methodologies and experimental
design is imperative to allow comparison of essential oil efficacy between studies.
Furthermore, the results from in vitro studies cannot be extrapolated into the field as
environmental factors can influence the biodegradation and volatility of essential oils and hence their
efficacy (Turek and Stintzing, 2013). The majority of in vivo studies use a topical application of essential
oil formulations to livestock as sprays or washes and then compare the number of flies found on the
treated and control individuals (Khater et al., 2009; Lachance and Grange, 2014). With all of these
protocols, it is imperative that appropriate controls are used. In addition to a synthetic insecticide
positive control, an excipient only and untreated negative control should be included. Furthermore,
in topical application and immersion experiments, using a non-essential oil as an additional positive
control is critical to allow the neurotoxic effect of an essential oil to be distinguished from the
mechanical effect of oils per se (Ellse and Wall, 2014). Without the use of appropriate controls any
effects observed cannot be attributed to the essential oil or compared to conventional treatments.
1.5.2 The use of essential oils against stable flies
Hieu and colleagues (2010a, 2010b, 2014) performed some of the first experiments
investigating the use of essential oils as potential repellents against stable flies. Hieu et al. (2010b)
assessed the repellent properties of 21 essential oils against stable flies using skin bioassays. Six
human volunteers had 12.5 mg of pure essential oil, diluted in ethanol, applied to the back of their
hands, at 0.5 mg/cm2 and were subsequently exposed to 15 female stable flies. Essential oils from
patchouli, Pogostemon cablin, clove bud, Eugenia caryophyllata and lovage root, Levisticum officinale,
showed the greatest potential as repellents as they protected subjects from stable fly bites for 3.67,
27
3.50 and 3.36 h, respectively (Hieu et al., 2010b). Furthermore, mixtures of essential oils with tamanu,
Calophyllum inophyllum, essential oil (0.25:2.0 mg/cm2), provided elongated protection times (Hieu
et al., 2010b). Alone leverage root and tamanu oil provided protection for 1.13 and 0.56 h,
respectively, whereas combined, this increased to 2.68 h, which exceeded the protection provided by
DEET (2.20 h). Using the same methods, essential oils obtained from Zanthoxylum piperitum and
Zanthoxylum armatum were assessed for their repellent properties (Hieu et al., 2010a, 2014). At 0.4
mg/cm2 Z. piperitum and Z. armatum treatments prevented 72% and 52% of stable flies from feeding
for 90 min. However, this was significantly lower than the positive control DEET which maintained
100% repellency over this period (Hieu et al., 2010b).
Citronella, Cymbopogon citratus, has also been assessed for its repellent properties against
stable flies (Baldacchino et al., 2013; Mottet et al., 2018). In electroantennogram experiments,
citronella initiated a strong response in stable flies, suggesting a behavioural response (Baldacchino
et al., 2013). In an experimental arena, the flight behaviour of stable flies, which were simultaneously
exposed to one essential oil impregnated (0.1 mg/μL) blood-soaked feminine hygiene pad and one
treated with hexane only (100 μL), were recorded over a 10 min observation period. Flies spent
significantly more time around the untreated blood source, with nine individuals (37.5%) taking a
blood meal. No flies fed on the citronella oil treated blood source (Baldacchino et al., 2013).
Furthermore, the authors recorded an overall decrease in the movement of flies over the observation
time. However, no trial was performed with two untreated blood sources to determine the natural
behaviour of these flies, therefore, the cause of reduced movement is uncertain.
Following this in vitro study, Mottet et al. (2018) investigated whether a citronella-based
formula could reduce fly annoyance behaviours in horses in vivo. The formulation consisted of
citronella oil (30 mL), distilled white vinegar (355 mL) and Avon Skin So-Soft® (118 mL), and when
sprayed on the legs of horses significantly reduced the number of tail swishes and shoulder twitches
performed per minute. Interestingly, pyrethrin spray (5%) did not significantly reduce these
behaviours, suggesting the citronella formulation was more effective (Mottet et al., 2018). However,
the only control in the study was an untreated horse, thus the reduction in fly annoyance behaviours
cannot be solely attributed to the presence of citronella due to the other components in the solution.
Therefore, despite the commercial utilisation of citronella based essential oil products, there is limited
evidence to support their use.
28
The most extensively studied essential oil against stable flies is catnip, Nepeta cataria, a
herbaceous mint plant (Zhu et al., 2009). The oil of catnip is rich in monoterpenoid nepetalactones,
which have been documented for their bioactivity against numerous insects (Peterson et al., 2002;
Bernier et al., 2005; Feaster et al., 2009). Firstly, Zhu et al. (2009) concluded that the oil was safe as
the results of broad-spectrum safety profiling were comparable to other Environmental Protection
Agency approved repellents. However, when applied topically (0.5 mL of pure oil) to New Zealand
white rabbits, all four subjects showed erythema within four days which persisted for the duration of
the experiment. Therefore, despite being categorised as a safe oil, the skin irritant properties of catnip
should not be overlooked. Subsequently, the insecticidal and repellent properties of catnip essential
oil against stable flies were investigated. The topical application of catnip oil concentrations of 50
μg/μL achieved 100% mortality in adult stable flies, although concentrations below 12.5 μg/μL, caused
negligible toxicity (Zhu et al., 2011). This dose-dependent response was also evidenced in fumigant
bioassays, as exposure to 100 μg/μL of catnip essential oil caused over 95% mortality whereas less
than 20% mortality was observed in 10 μg/μL treatments (Zhu et al., 2011). This study is valuable as
the insecticidal effects of catnip against stable flies is investigated, whereas the majority of studies
focus on their repellent properties alone.
In in vitro repellency bioassays, Zhu et al. (2009) showed that impregnating the membranes
of citrated bovine blood-soaked feminine hygiene pads with 300 μL of 67 μg/μL of catnip oil prevented
96% of starved stable flies from feeding for 4 h. However, at lower concentrations of 6.7 μg/μL and
0.67 μg/μL, no significant repellent effect was recorded. Interestingly, subsequent experiments
showed that 70% of flies engorged when presented with a food source treated with 0.67 μg/μL of
catnip oil, a percentage comparable to the mineral oil control (Zhu et al., 2012). Catnip oil has also
been exemplified as a strong deterrence of oviposition as a catnip-treated barrier (0.1 g/mL) around
oviposition media repelled 98% of gravid females for 6 h (Zhu et al., 2012). Collectively, these
experiments show the contact and spatial repellent properties of catnip oil when administered at
higher concentrations.
The efficacy of catnip oil was examined in vivo in field trails where the application of 250 mL
of 30% (v/v) water-based and 15% (v/v) oil-based catnip essential oil formulation onto the legs of
cattle significantly lowered the number of residing flies for 5 and 6 h, respectively (Zhu et al., 2012).
However, it must be noted that the mineral oil control also had a significant repellent effect on stable
flies, thus the efficacy of the oil-based formulation cannot be solely accredited to the essential oil
component (Zhu et al., 2012). Furthermore, in each trial, one front and one hind leg was treated with
29
the essential oil and the other was the control. However, due to the evident spatial repellency of
catnip oil, the controls are not independent of the effects of the essential oil and thus are not an
appropriate comparison. Despite these shortcomings in the experimental design of this study, the
results suggested that even at high doses the in vivo repellent effect provided by catnip oil is short-
lived. This is problematic as high doses are likely to be expensive and have associated safety concerns,
especially considering the irritability of this oil to skin.
To try and increase longevity and efficacy, Zhu et al. (2014) encapsulated catnip essential oil.
The capsules were composed from a pork skin gelatine wall matrix and a core which consisted of pure
essential oil and mineral oil (1:1) (Zhu et al., 2014). Oviposition media coated with 0.5 g of
microencapsulated catnip contained 98% fewer eggs than the control. However, this effect
disappeared within 48 h. In growth inhibition assays, where stable fly eggs were placed on a
developmental media treated with 0.5 g of these gelatine microcapsules, only 0.6% of third-stage
larvae matured and survived after 7 d (Zhu et al., 2014). It was noted that the media treated with
catnip oil contained significantly fewer microbial communities and it was hypothesised that the
inhibition of larval growth may be a consequence of decreased food resources for larvae (Zhu et al.,
2014).
In conclusion, few essential oils have been examined extensively for their repellent efficacy
against stable flies and even fewer for their insecticidal effects. Of those which have, there are often
been limitations in the experimental design. Catnip essential oil has been the most extensively studied
but its practical use in the field has not yet been demonstrated clearly.
1.6. Aim of this thesis
The overall aim of the work described here was to assess the insecticidal and repellent efficacy
of essential oils against stable flies. The first aim was to select potentially valuable essential oils
through a semi-quantitative literature search, based on studies which had examined efficacy against
other biting flies. Then, in vitro bioassays were to be designed to investigate the toxicity of selected
essential oils. Subsequently, the aim was to use behavioural bioassays to explore the repellent quality
of the chosen essential oils.
30
Chapter 2
Insecticidal and repellent effects of lavender,
Lavandula angustifolia, and tea tree, Melaleuca
alternifolia, essential oils against stable flies
2.1 Introduction
Stable flies are important ectoparasites due to their ubiquitous distribution and close
association with economically valuable livestock hosts (Foil and Hogsette, 1994). The most common
approach to their control is through the use of synthetic pesticides such as organophosphates and
pyrethroids (Muraleedharan, 2005; Mottet et al., 2018). However, recently, the negative
consequences and diminishing effectiveness of these conventional treatments has become evident
and there has been growing interest in finding sustainable alternative control mechanisms, including
plant-based repellents and pesticides. Repellents could be used as components of an integrated pest
management approach in conjunction with improved sanitation and removal of oviposition site
material (Hogsette et al., 1987; Holdsworth et al., 2006).
The first aim of this study, therefore, was to semi-quantitively assess the efficacy of essential oils
previously tested on biting flies and determine which held the greatest potential. Based on this
assessment, two essential oils would be chosen for further investigation to determine their insecticidal
and repellent properties against stable flies. It was hoped that the results from this study would
contribute towards our understanding of essential oils as stable fly control agents and assist in the
development of formulations which could be used against a range of ectoparasites in the field.
2.2 Methods and materials
2.2.1 Stomoxys calcitrans
A stable fly colony was established at the University of Bristol using pupae obtained from a
30-year old laboratory colony maintained at MSD Animal Health Innovation (Schwabenheim,
Germany). The flies were maintained in entomological cages (30 x 30 x 30cm) at 22 ±0.5°C with 40–
45% relative humidity under a 18:6 light:dark photoscopic period. Adult flies were fed daily by placing
4 g cotton wool soaked in 5mL citrated bovine blood (100 mL of 4% sodium citrate/L) in their cage.
31
Blood was collected regularly from the School of Veterinary Science abattoir (Langford, Bristol).
Bioassays were conducted under the same laboratory conditions as used to maintain the flies.
2.2.2. Essential Oils
A semi-quantitative analysis of primary literature evaluating the efficacy of essential oils
against biting flies was conducted to determine which essential oils had the greatest potential. The
relevant peer-reviewed literature was found by searching on Web of Science (v.5.31; 15.10.2019;
https://clarivate.com/products/web-of-science/) using the key terms: “essential oil” or “extract” or
“plant product” and “insecticidal” or “repellent” and “Ceratopogonidea” or “Simulidae” or
“Tabanidae” or “Muscidae” or “Psychodidae” or “Glossinidea”. A database of 31 studies investigating
68 essential oils was compiled, and points were allocated to the oils based on the following criteria:
repellent and/or insecticidal efficacy; concentration of essential oil; experimental design (controls,
sample size and methods); and practicality (cost, availability, safety). Each criterion was scored out of
five points. To determine the consistency of essential oils, the mean number of points allocated to
each essential oil was calculated. Based on this analysis, lavender, Lavandula angustifolia and tea tree,
Melaleuca alternifolia, essential oils were selected for further investigation.
Steam-distilled lavender and tea tree (100%) essential oils were obtained from a commercial
source (Naissance Trading and Innovation, Neath, UK). To prevent thermo-degradation these essential
oils were maintained at 5±1°C in complete darkness (Najafian, 2016). To achieve 5% (v/v)
concentrations, the essential oils were diluted with absolute ethanol (≥ 99.8%; VWR international,
France). Furthermore, absolute ethanol was used as negative control to distinguish effects caused by
the excipient. DEET (20% v/v) (97%; Sigma-Aldrich, Gillingham, UK), diluted in absolute ethanol, was
used as a positive control. For each experimental treatment and replication, fresh suspensions of
essential oils were made to avoid concentration and composition differences caused by evaporation
and biodegradation.
2.2.3 Insecticidal Bioassay
The insecticidal effect of each oil was examined using filter papers impregnated with the test
formulations, in an adaptation of the WHO insecticide resistance protocol (Farnsworth et al., 1997;
Cossetin et al., 2018). First, filter papers (Whatman No. 1; 150mm diameter) were fully saturated with
a 1mL aliquot of each treatment: 5% (v/v) lavender essential oil; 5% (v/v) tea tree essential oil;
absolute ethanol (excipient only control). This produced a concentration of 0.283 𝜇L/cm2 of essential
oil on the filter paper. Filter papers spent 5 min in a fume cupboard to allow the solvent to evaporate
32
before being placed into a 135mm diameter plastic Petri-dish. Simultaneously, one-week old stable
flies, of mixed sex, were briefly chilled (-14°C) to inactivity, and ten randomly chosen flies were placed
on each of the dry filter papers and the Petri-dish lid was secured in place. Live and dead flies were
counted over a 2-min observation period at 15, 30, and 45 min and 1, 2, 4, 6 and 24 h post exposure.
A fly was recorded as dead if no movement was detected during the 2-min observation time and no
response was detected after agitation with a paintbrush. All tests were performed in triplicate, using
10 new flies and formulations for each replication.
2.2.4 Repellency Bioassay
A repellency bioassay was designed to determine whether essential oils could deter stable
flies from feeding (Fig. 2.1). Pre-experimental observations indicated that one-week old stable flies
generally alighted on the upper surface of their cage and hence the test apparatus was designed
accordingly.
An olfactometer apparatus consisted of a 40 cm long vertical tube, constructed from plastic
beakers and drinks bottle. A feeding attractant composed of 4 g of cotton wool soaked in 5 mL of
citrated bovine blood, was placed on a mesh-ended plastic cup, which formed the feeding chamber
at the top of the apparatus (Fig. 2.1). Immediately below the attractant was a funnel constructed using
the neck from a 2 L plastic drinks bottle. The funnel was lined with filter paper (Whatman No. 1). The
filter paper had a 2 cm diameter central circular hole to allow the movement of flies through the
apparatus to the attractant. Immediately prior to a test, the filter papers were saturated with 1 mL of
a test solution: 5% (v/v) lavender essential oil, 5% (v/v) tea tree essential oil, 20% (v/v) DEET (positive
control), absolute ethanol (excipient only) or no treatment. Filter papers were then placed into a fume
cupboard for 5 min to allow the ethanol solvent to evaporate and were then secured into the bottle
neck. An airflow through the apparatus from top to bottom was created using an electric fan (5 V DC,
25x25x10mm, 5.95 m³/h, 600 mW, Sunon LTD, Kaohsiung City, Taiwan) powered by a 6 V DC battery.
One-week-old stable flies, which had been starved for 24 h, were briefly chilled (-14°C) and
randomly allocated into mixed-sex groups of 10 and assigned a treatment were then placed in the
lower chamber of the apparatus. This was composed of a mesh-ended plastic cup (Fig. 2.1). To obtain
a blood meal, flies would have to travel from the lower chamber, through a treated funnel, into the
upper feeding chamber. Preliminary experiments, with untreated filter papers, had shown that usually
all stable flies had reached the blood-soaked cotton wool and fed within 60 min; thus, this would be
an appropriate length of time to determine if test formulations affected the feeding behaviour of
33
stable flies. Once the flies had been introduced into the apparatus, the number of flies which had
passed through the treated tunnel into the upper feeding chamber were counted at 5, 15, 30, 45 and
60 min.
Figure 2.1. The experimental apparatus
used to determine if essential oils were a
feeding deterrent to Stomoxys calcitrans.
(1) Blood soaked cotton wool placed on (2)
a mesh-ended plastic pint cup which
formed the upper feeding chamber. (3) The
funnel was constructed from a 2L plastic
bottle neck containing a treated filter
paper. (4) A plastic pint cup connected to
(5) a half-pint plastic cup with a mesh
bottom which formed the entrance
chamber. (6) Electric fan for airflow
through the apparatus.
2
3
4
5
6
1
34
2.2.5 Statistical Analysis
All statistical tests were performed using RStudio (R Core Team, R Foundation for Statistical
Computing, Vienna, Austria, Version 3.6.3, 2020), and a difference was considered statistically
significant if P<0.05. Firstly, data was tested for homogeneity using Shapiro-Wilk test and normality of
variance using a Levene’s test. Both data sets were normally distributed and thus analysis of variances
(ANOVA) were performed, followed by a Tukey post-hoc tests to determine differences between
groups. For the insecticidal bioassay, the number of dead flies 15 min post-exposure was the response
variable and treatment as the independent variable. This time frame was considered for analysis
because if the essential oils were to be used in the field, they would need to be effective after a short
period of exposure. In the repellency bioassays, the number of stable flies that reached the feeding
chamber at 60 min was the response variable, with treatment as the independent variable. In each
bioassay, recordings made at a single time only were used for ANOVA, to prevent the problems
associated with non-independent observations. The time taken to achieve 50% mortality (LT50) post
exposure to treatment in insecticidal bioassays was determined for both lavender and tea tree
essential oils using the dose.p function in RStudio.
2.3 Results
2.3.1 Essential Oils
From the literature search, 31 studies investigating the efficacy of 68 essential oils on over 15
species of biting flies were found (Appendix I). The plant family Lamiaceae had the highest
representation (32.31%), followed by Asteraceae (13.85%), Myrtaceae (12.31%) and Rutaceae
(7.69%). The essential oil most frequently tested was rosemary, Rosmarinus officinalis (5), followed
by catnip (4), lavender (4) and tea tree (4).
35
Table 2. The number of points allocated to the top five performing essential oils.
Plant Species (Common name)
Average Number of Points Allocated to Each Category Total Score
Efficacy Concentration Experimental
Design Practicality
Melaleuca alternifolia (Tea tree)
4 3.75 4.25 5 17
Lavandula angustifolia (Lavender)
3.75 3.75 3.5 5 16
Carapa guianensis (Andiroba)
3.5 3.5 5 3 15
Pelargonium graveolens (Pelargonium)
3.5 3.5 3.5 3 15
Nepeta cataria (Catnip)
3.5 3 3.75 4 14.25
2.3.2 Insecticidal Bioassay
In the analysis of the insecticidal efficacy of essential oils, even after only 15 min, stable fly
mortality significantly varied between treatments (ANOVA, F4=19, P<0.001); significantly more stable
flies died after exposure to filter papers impregnated with 5% (v/v) lavender essential oil (Tukey HSD,
p<0.05) and 5% (v/v) tea tree essential oil (Tukey HSD, P<0.001), compared to the excipient-only
ethanol controls (Fig 2.2). However, the mortality caused by exposure to lavender or tea tree essential
oil was not significantly different (Tukey HSD, P=0.64). On average 3 ±0.58 and 3.67 ±0.67 flies died
when exposed to lavender and tea tree oils, respectively, whereas no flies died when exposed to the
ethanol control for 15 min (Fig. 2.3). The LT50 for lavender and tea tree essential oils were 54 and 51
min, respectively. Lavender essential oil caused 100% stable fly mortality within 4 h and tea tree within
6 h.
2.3.4 Repellency Bioassay
After 60 min in the apparatus the number of stable flies that reached the end chamber
containing the blood varied significantly (ANOVA, F4=19, P<0.001). Subsequent multiple comparison
tests showed that the number of flies that passed the filter papers impregnated with lavender and tea
tree essential oils were significantly less than untreated controls (Tukey HSD, P<0.001) and excipient-
only ethanol controls (Tukey HSD, P<0.01). After 60 min, when exposed to untreated or excipient-
treated filter paper funnels, a mean ± standard error of 9.67 ±0.33 and 7.67 ±0.33 flies passed into the
feeding chamber, respectively, whereas only 1.67 ±1.67 and 1 ±0.58 flies had done so when the filter
papers were impregnated with 5% lavender or tea tree essential oil, respectively. Only 3.67 ±0.67 flies
36
passed the filter paper when impregnated with DEET. The number of flies that passed the filter papers
impregnated with the lavender (Tukey HSD, P>0.05) or tea tree (Tukey HSD, P>0.05) oils compared to
the positive control DEET was not significantly different. However, the number of flies that passed the
DEET impregnated filter papers was not significantly different from that observed with the ethanol
negative controls (Tukey HSD, P>0.05).
Figure 2.2 Mortality (mean ±SE) of Stomoxys calcitran at 15, 30 and 45 min and 1, 2, 4, and 6 h post-
exposure to filter papers impregnated with 5% (v/v) lavender essential oil (○), 5% (v/v) tea tree
essential oil (▲) and absolute ethanol excipient-only negative control (■). Points have been offset and
joined for clarity.
0
1
2
3
4
5
6
7
8
9
10
0 1 2 3 4 5 6
Mea
n s
tab
le f
ly m
ort
alit
y
Exposure time, h
37
Figure 2.3 The number of Stomoxys calcitrans (mean ±SE) that reached the end chamber of an
olfactometer containing blood-soaked cotton wool after passing a filter paper funnel impregnated
with 5% (v/v) lavender essential oil (○), 5% (v/v) tea tree essential oil (▲), DEET (20% v/v) positive
control (●), absolute ethanol excipient-only negative control (□) and untreated negative control (■) at
baseline, 5, 15, 30, 45 and 60 min. Points have been offset and joined for clarity.
2.4 Discussion
0
1
2
3
4
5
6
7
8
9
10
0 15 30 45 60
Mea
n n
um
ber
of
stab
le f
lies
fee
din
g
Exposure time, min
38
Exposure to filter papers impregnated with either 5% lavender or tea tree essential oil caused
significantly greater stable fly mortality than the control, even within 15 min, and achieved 100%
stable fly mortality within 4 and 6 h, respectively (Fig. 2.2). The very low mortality in the excipient only
controls, even after 24 h, provides a high degree of confidence that this mortality was a consequence
of the essential oils (Fig. 2.2). The toxicity observed here appears to be greater than that recorded for
catnip; when exposed to catnip oil, even at higher doses (0.2 mg/μL), 100% stable fly mortality was
not achieved and 20% of flies originally recorded as dead recovered (Zhu et al., 2011). Here, doses
equivalent to ~0.044 mg/μL were sufficient to give 100% mortality and recovery was not observed
(Naissance, 2020). The rapid knockdown and mortality achieved by lavender and tea tree oils shows
their insecticidal potential against stable flies.
The results of this study are in accordance with previous biting fly experiments. Using a similar
bioassay, Cossetin et al. (2018) showed the efficacy of lavender oil on the calliphorid Chrysomya
albiceps, as concentrations of 0.1 mg/cm2 caused 100% mortality within 2 h. Similarly, in adaptations
of the CDC bottle bioassay, 0.2% (v/v) of lavender oil was shown to cause 100% mortality of Lucilia
sericata within 5 min (Khater and Geden, 2018). Tea tree has also been shown to be toxic to biting
flies; the topical application of 1% (v/v) tea tree oil killed 100% of horn flies, Haematobia irritans,
within 3 h and, in feeding assays, 2.5% (v/v) concentrations caused 100% mortality of Lucilia cuprina
second stage larvae (Callander and James, 2012; Klauck et al., 2014). The variation in concentrations
required to achieve mortality in different species may be a consequence of interspecific variation in
fly size, pilosity, and physiological susceptibility and differences in experimental design.
The insecticidal efficacy and mode of action of essentials oils can be influenced by the
experimental design of the study. For example, the fumigant toxicity of essential oils is associated with
their vapour pressure and this can be influenced by the excipient and experimental arena used (Ajayi
et al., 2014). Here, essential oils were mixed with very volatile ethanol and the Petri-dishes formed
small closed chambers, thus flies would have been exposed to high concentrations of oils in their
vapour phase (Sfara et al., 2009; Koutsaviti et al., 2018). Previous studies have shown that the
effectiveness and residual activity of oils is greater in closed chambers compared to open ones
(George et al., 2008; Sands et al., 2016). Therefore, the mortality observed here can be mainly
attributed to the absorption and inhalation of essential oils in their vapour phase (Cossetin et al.,
2018). Analysis in an open environment would be an appropriate next step to evaluate their effect in
the field.
39
The equally rapid adulticidal effect caused by exposure to lavender and tea tree essential oils here
is likely to have been due to their high concentrations of oxygenated compounds. Lavender contains
a high proportion of linalyl acetate (43-48%), linalool (28%-34%) and 1,8-cineole (18%-24%) and tea
tree oil consists of high proportions of terpinen-4-ol (35-48%) and 1,8-cineole (10%) (Najafian, 2016;
ISO, 2017). Papachristos and colleagues (2004) found that essential oils containing higher proportions
of oxygenated monoterpenoids exhibited increased insecticidal activity against the bean weevil,
Acanthoscelides obtectus, thus this shared characteristic can help explain the effectiveness of lavender
and tea tree oil. More specifically, these monoterpenoid compounds have been found to interfere
with insect acetylcholinesterase and GABA receptors and result in the deregulation of the
neuromuscular system, ataxia and insect death (Table 1). One of the most powerful inhibitors, 1,8-
cineole, can cause 50% inhibition of acetylcholinesterase at doses as low as 0.015 mg/mL (Dohi et al.,
2009). Furthermore, minor components of these oils act synergistically to improve efficacy. For
example, α-Pinene, a minor component of lavender (2.3%), is thought to work synergistically with 1,8-
cineole to increase inhibition of acetylcholinesterase (Savelev et al., 2003; Najafian, 2016).
Collectively, the major and minor components of these oils are responsible for their efficacy.
Both lavender and tea tree essential oils prevented flies from passing impregnated filter papers
within the olfactometer, and this suggests that both show promise as botanical stable fly repellents.
Both oils were able to deter more flies from the food source than 20% DEET, a commercially available
repellent recommended by the WHO to be used as a positive control when assessing new repellents
(WHO, 2009). Furthermore, tea tree consistently repelled more flies from the feeding chamber than
lavender oil, and with lower variation. Both of these oils showed great potential as botanical
repellents. By definition, botanical repellents are natural substances which stimulate an avoidance
response from their target species (Zhu et al., 2015). This can be further categorised as either contact
or spatial repellents, whereby contact repellents cause adverse reactions in target species post
contact, and spatial repellents work in their vapour phase as volatile components are detected by the
insect’s olfactory sensilla and initiate an avoidance behaviour before contact (Achee et al., 2009).
Here, stable flies were observed flying away from the treated filter papers, before contact was made,
implying a spatial repellent effect. Furthermore, the design of the bioassay used here suggests that
the repellent behaviour was not likely to have been associated with an adverse reaction to the
ingestion of the essential oil (Zhu et al., 2012). Spatial repellence is a particularly useful quality in the
field as a treatment could prevent stable flies coming in close proximity to hosts and hence avoid
defensive host behaviours and the consequences associated with them (Section 1.2). Further
40
investigation of the spatial repellent properties of these oils, using electroantennogram analysis, may
be of value (Hieu et al., 2014).
In comparison to other essential oils used against stable flies, lavender and tea tree show greater
promise as practical botanical pesticides, not only because of their great efficacy but also because
these oils are considerably less expensive (Hieu et al., 2010; Zhu et al., 2011; Naissance, 2020). What
is more, not only are these oils effect against stable flies, their repellent qualities have been evidenced
against multiple biting fly species. For example, 1 μg/μL of lavender oil in hexane repelled 93.7% of
flies for 4 min (Gonzalez et al., 2014). Furthermore, through a series of in vitro and in vivo experiments,
formulations of tea tree oil (5% (v/v)) have successfully repelled horn flies from feeding for up to 24 h
(Klauck et al., 2014, 2015). Tea tree oil (3% (v/v)) also prevented oviposition in L. cuprina for 6 weeks
(Callander and James, 2012). Encouragingly, all of the doses are low and thus a treatment of 5%
lavender or tea tree appears likely to be an appropriate field dose to facilitate insecticidal and
repellent effects against a variety of biting flies.
The final aim of the work to be undertaken as part of this research project was to conduct an
investigation of the efficacy of these oils when applied to donkeys in the field at the Donkey Sanctuary
farm in Devon. However, due to the COVID-19 outbreak this work was not possible. In the field,
variable temperatures, ultraviolet light, wind and rain, may all increase the biodegradation of essential
oils and reduce their efficacy and residual activity (Turek and Stintzing, 2012). Nevertheless, the
repellent qualities of these oils have been examined in the field against other fly species. For example,
the application of 5% (v/v) concentrations of lavender and tea tree significantly reduced the number
of flies alighting on pastured cows for up to 5 and 24 h, respectively (Klauck et al., 2014; Lanchance
and Grange, 2014). Consequently, it can be concluded that both lavender and tea tree show promise
as effective and sustainable control strategies against stable flies, and therefore they warrant further
in vivo investigation to fully elucidate their potential in the field.
41
Chapter 3
General Discussion
3.1. General Discussion
Lavender and tea tree belong to the Lamiaceae and Myrtaceae family, respectively, which are
among the most widely studied families for their pharmacological properties (Benelli and Pavela,
2018a). Lavender is an aromatic shrub native to the Mediterranean and the flowers produce a
colourless oil with a strong floral fragrance (Cavanagh and Wilkinson, 2002). Comparatively, tea tree
oil is usually pale yellow in colour with a distinct camphoraceous odour and is extracted from the tea
tree plant which is native to East Australia (IOS, 2017). Both of these oils have been used as
ethnobotanical therapeutic agents for centuries and with the increasing popularity in botanical
alternatives, they have become of great interest over the last few decades (Cavanagh and Wilkinson,
2002; Yadav et al., 2017). In Chapter 2, the insecticidal and repellent properties of lavender and tea
tree essential oils were demonstrated against stable flies and both showed promise as natural
alternatives to conventional synthetic insecticides.
For the use of these oil-based treatments in animal husbandry, they must be effective, safe,
easily applied and economically viable. Synthetic neurotoxins are usually effective against a broad
range of ectoparasites and hence, if essential oils are going to be considered as a viable alternative to
synthetic pesticides, they must provide protection against a similar range of target species (Campbell,
1985). Encouragingly, the results from numerous field and laboratory experiments have shown
efficacy of both lavender and tea tree against an array of veterinary important ectoparasites, including
flies (see Appendix I) lice (James and Callander, 2012; Ellse et al., 2015), mites (Mägi et al., 2006) and
ticks (Perino-Issartier et al., 2010; Pazinato et al., 2014). For example, Ellse and colleagues (2016)
showed that two weeks after hand spraying donkeys with a 5% (v/v) lavender and tea tree
formulation, the number of Bovicola ocellatus found on treated individuals decreased by 78%.
Similarly, Mägi and colleagues (2006) showed that four weeks after treating pigs with 1% (v/v) tea tree
emulsions, their sarcoptic mange mite, Sarcoptes scabiei, intensity of infection decreased by over 98%.
The broad-spectrum efficacy of lavender and tea tree means they are likely to be viable alternatives
to conventional neurotoxic treatments. Additionally, it is unlikely that an insect will acquire resistance
to essential oil treatments due to their complex modes of action (see section 1.4). This is not only
beneficial for the long-term efficacy of the treatment, but it also means that unlike with conventional
42
treatments, where targeted application is required to reduce the risk of resistance, essential oils could
provide a year-round, long-term solution to prevent infestations from several parasites.
Lavender and tea tree essential oil also have antibacterial and fungicidal properties which
could make them advantageous over conventional neurotoxins. Previous work has shown that
concentrations of 0.013% and 0.5% (v/v), of lavender and tea tree oil, can initiate bacterial cell death
(Cox et al., 2000, 2001; Sienkiewicz et al., 2014). More specifically, the lipophilic monoterpenoid
components of these oils can affect the structural and functional properties of a bacterial membrane
and consequently cause the dysregulation of intercellular homeostasis and inhibit cell respiration
(Sikkema et al., 1995; Cox et al., 2000, 2001). Furthermore, terpinen-4-ol, linalool and 1,8-cineole have
been shown to be effective fungicides at concentrations below 0.25% (Hammer et al., 2003). As these
compounds are highly represented in lavender and tea tree oils, they too could have useful fungicidal
effects. Therefore, the topical application of these oil formulations may not only reduce ectoparasite
numbers but could also improve the dermal health of the treated animal. What is more, these
pharmacological properties have been demonstrated at low doses, thus 5% concentrations would be
likely to provide adequate control against a wide range of ectoparasites, bacteria and fungi.
Lower concentrations of essential oils are beneficial as they are associated with minimised
safety concerns. This is of particular interest if formulations are to be topically applied to animals
which partake in self-grooming activities. Previous work has shown no skin irritability when 5%
concentrations of lavender and tea tree essential oils have been topically applied to livestock and
companion animals (Lachance and Grange 2014; Klauck et al., 2014; Ellse et al., 2016). Furthermore,
the oral toxicities of lavender (LD50: >2 g/kg) and tea tree oils (LD50: 1.9–2.6 ml/kg) are below that of
conventional insecticides (Russell, 1999; Mekonnen et al., 2019; Cantalamessa, 1993). However, due
to the lipophilic nature of essential oils, transdermal absorption can occur and thus residues of oils
may accumulate in the muscles of treated animals (Herman and Herman, 2014). At present, no work
has been conducted to elucidate the potential tainting of animal products, such as milk and meat,
when oils are topically administered. However, it is unlikely to be a significant issue as Rivaroli et al.
(2016) showed that the inclusion 3 g/animals/day of essential oil blends into the feeding regime of
crossbred bulls had no effect on the chemical composition of their meat, thus tainting is improbable
but specific analysis into the effect of topical application is required. Consequently, at present, these
oil treatments can only be safely recommended for use on companion animals. Future work can focus
on the commercialisation of impregnated tail tags for cattle, although it is likely these will only be
43
effective against flies and not provide simultaneous protection against permanent parasites (Hogsette
et al., 1987; Juan et al., 2011).
Within companion animals, equids are readily attacked by stable flies and are hosts to
numerous other ectoparasites, thus are prime candidates for these botanical treatments (Patra et al.,
2018; Karasek et al., 2020). Several botanical treatments have already been commercialised for this
market and hence there is an acceptance for these products within this sector which could be utilised.
The essential oil formulations could be applied using the hand spray method employed by Ellse et al.
(2013, 2016) for the treatment of donkeys. In the latter study, 2 mL of essential oil formulation was to
be applied per kg body weight (to the nearest 50 kg) of the animal. Therefore, for an average size
donkey, 400 mL of the solution was to be sprayed onto the individual during routine grooming
practices. This simple and convenient spray technique is analogous to current treatments and hence
could easily be introduced as an alternative. Furthermore, similar application methods have been
shown to be operational on other animals which are targeted by stable flies, such as dogs (Goode et
al., 2018).
In terms of costs, the extraction of essential oils from aromatic plants is an expensive process
due to the specific equipment required for distillation and the low oil yields (0.5-6.8%) obtained from
plant material (Zheljazkov et al., 2013). Fortunately, due to the popularity of lavender and tea tree
essential oil in the food, cosmetic and natural health industries, they are commercially produced and
hence are among the most affordable oils (Naissance, 2020). If the same application methods as Ellse
et al. (2016) were used, based on current trading prices and already commercialised products, it would
cost between £6.40 and £20 per treatment per animal, depending on excipients used (Agrient Limited,
2020; NAF UK, 2020). For high value animals such as equids, this is comparable to many of the
conventional synthetic treatments used (e.g. Tri-Tec 14™, Farnam and NAF-Off DEET Power
Performance, NAF). However, these costs would be inhibitory for use on livestock due to the increase
in scale of use. The principle of using endemic botanical-based pesticides may be particularly attractive
in less economically lucrative countries, as endemic plant species can provide a sustainable alternative
to high cost synthetics, but at present this is not possible. Therefore, to allow the use of essential oil-
based products on a greater range of animals and globally, there should be a continued focus on
improving the oil yield through biotechnology and reducing the cost of the extraction process.
Isman (2006) has argued that the limited residual activities of essential oil-based formulations
could inhibit their commercialisation. Their short period of effectiveness is less problematic in the
44
control of permanent ectoparasites, such as B. ocellatus, as one treatment could eliminate an entire
parasite population if hosts are treated simultaneously and the risk of immigration was minimal (Ellse
et al., 2015; Sands et al., 2016). However, to afford continuous protection against parasites with free-
living stages, such as stable flies, a prolonged efficacy is fundamental. Therefore, if only effective over
a short period, higher application frequencies may be required, and this may result in annual costs
exceeding that of conventional treatments. However, in equid husbandry, numerous synthetic
treatments require daily application including DEET, and hence the short residual activities of essential
oils may be less problematic (Herholz et al., 2016). In the present study, both tea tree and lavender
significantly deterred starved stable flies from a blood source for one hour. However, due to the
laboratory conditions and short time frame this data cannot be used to estimate their residual activity
in the field. To more effectively quantify the residual activity of these essential oils in the field, in vivo
studies, whereby the oils are applied to the animal hide, must be performed.
The excipient used for essential oil application can have a profound effect on the efficacy and
residual activity of the treatment. Firstly, the design of the formulation can alter the hydrophobicity
and improve its penetration into the coat of the animal and hence its residual activity. For example,
James and Callander (2012) assessed the efficacy of tea tree oil against B. ovis on sheep and showed
that the excipient used, which consisted of water, oleic acid and ethoxylated castor oil, assisted in the
penetration of the essential oil into the wool. The authors also claimed that even after several weeks,
the tea tree odour could be detected; this prolonged period of activity would be beneficial when an
animal is under repeated challenge from stable flies. Furthermore, previous work has shown that
different excipients can have a significant effect on the transdermal penetration of drugs and their
distribution throughout the skin (Mills et al., 2005; Mills, 2007). Therefore, future work should
continue to investigate the effectiveness of essential oils in combination with different excipients in
vivo as this may help improve their residual activities and prevent transdermal absorption and hence
assist in their commercialisation in different sectors.
Despite the research into essential oils and their pesticidal properties, commercialisation of such
formulations is limited. Before their use as medicines, these bioinsecticides require regulatory
approval. In several countries, botanical pesticides are not distinguishable from conventional
treatments and hence have to go through the same expensive regulatory processes (Isman, 2006).
Due to the smaller market and profit margins for botanical pesticides, the cost of this process of
registration could be inhibitory. However, the United States of America have exempted several
essential oils from registration due to their popularity in the cosmetic and food industry and hence
oil-based pesticides have been commercialised for over a decade. Similarly, the European Union has
45
excused essential oil-based formulation from registration if they are not for human use and
consequently, in the past six years there has been an increase in the availability of botanical pesticides
for companion animals and livestock (Isman, 2019). Therefore, due to their current acceptance in the
European Union, it is probable that a new essential oil product would be exempt from registration.
However, perhaps if this industry become lucrative and more products become available, or different
solutions are mixed with essential oils, the regulatory processes may change. The variation in
regulatory approval processes around the world is still a barrier to the commercialisation of essential
oil pesticides and hence an appropriate unanimous regulatory system needs to be established.
3.2 Conclusions
The experiments conducted as a part of this theses showed the high efficacy of 5% (v/v)
concentrations of lavender and tea tree essential oils as pesticides and repellents against stable flies.
Therefore, both oils are potential options as botanical alternatives to synthetic neurotoxic treatments
for the control of stable flies and can be used in conjunction with the removal of material conducive
to oviposition for an effective integrated pest management scheme. Furthermore, due to the broad
range of ectoparasitic species affected by lavender and tea tree oil, their topical administration to
animals may provide protection against a range of important pests. However, before these can be
advocated for use, there must be field trials to elucidate their efficacy and residual activity under field
conditions. It is likely these essential oil formulations can be incorporated into companion animal
husbandry practices, but further work is needed to extend the residual activity of these oils and
establish their safety before use on food production animals.
46
References
Achee, N.L., Sardelis, M.R., Dusfour, I., Chauhan, K.R. and Grieco, J.P. (2009) Characterization of
spatial repellent, contact irritant, and toxicant chemical actions of standard vector control
compounds. Journal of the American Mosquito Control Association, 25, 156–167.
Adenubi, O.T., Ahmed, A.S., Fasina, F.O., McGaw, L.J., Eloff, J.N. and Naidoo, V. (2018) Pesticidal
plants as a possible alternative to synthetic acaricides in tick control: A systematic review and
meta-analysis. Industrial Crops and Products, 123, 779–806.
Agee, H.R. and Patterson, R.S. (1983) Spectral sensitivity of stable, face, and horn flies and
behavioral responses of stable flies to visual traps (Diptera: Muscidae). Environmental
Entomology, 12, 1823–1828.
Agrient Limited (2020) Equine Nitnat. [online] Available at:
<https://www.agrientlimited.com/equine-nitnat> [Accessed 7 July 2020].
Aïzoun, N., Ossè, R., Azondekon, R., Alia, R., Oussou, O., Gnanguenon, V., Aikpon, R., Padonou, G.G.
and Akogbéto, M. (2013) Comparison of the standard WHO susceptibility tests and the CDC
bottle bioassay for the determination of insecticide susceptibility in malaria vectors and their
correlation with biochemical and molecular biology assays in Benin, West Africa. Parasites and
Vectors, 6, 1–10.
Ajayi, O.E., Appel, A.G. and Fadamiro, H.Y. (2014) Fumigation toxicity of essential oil monoterpenes
to Callosobruchus maculatus (Coleoptera: Chrysomelidae: Bruchinae). Journal of Insects, 2014,
1-7.
Anderson, J.A. and Coats, J.R. (2012) Acetylcholinesterase inhibition by nootkatone and carvacrol in
arthropods. Pesticide Biochemistry and Physiology, 102, 124–128.
Anderson, J.R. (1978) Mating behavior of Stomoxys calcitrans: effects of a blood meal on the mating
drive of males and its necessity as a prerequisite for proper insemination of females. Journal of
Economic Entomology, 71, 379–386.
Andersson, M.N., Löfstedt, C. and Newcomb, R.D., (2015) Insect olfaction and the evolution of
receptor tuning. Frontiers in Ecology and Evolution, 3, 1-53.
Andress, E.R. and Campbell, J.B. (1994) Inundative releases of pteromalid parasitoids (Hymenoptera:
Pteromalidae) for the control of stable flies, Stomoxys calcitrans (L.) (Diptera: Muscidae) at
confined cattle installations in west central Nebraska. Journal of Economic Entomology, 87,
714–722.
Angioni, A., Barra, A., Coroneo, V., Dessi, S. and Cabras, P. (2006) Chemical composition, seasonal
variability, and antifungal activity of Lavandula stoechas L. ssp. stoechas essential oils from
stem/leaves and Flowers. Journal of Agricultural and Food Chemistry, 54, 4364–4370.
47
Bakkali, F., Averbeck, S., Averbeck, D. and Idaomar, M. (2008) Biological effects of essential oils - A
review. Food and Chemical Toxicology, 46, 446–475.
Baldacchino, F., Tramut, C., Salem, A., Liénard, E., Delétré, E., Franc, M., Martin, T., Duvallet, G. and
Jay-Robert, P. (2013) The repellency of lemongrass oil against stable flies, tested using video
tracking. Parasite, 20, 1–7.
Baleba, S.B.S., Torto, B., Masiga, D., Getahun, M.N. and Weldon, C.W. (2020) Stable flies, Stomoxys
calcitrans L. (Diptera: Muscidae), improve offspring fitness by avoiding oviposition substrates
with competitors or parasites. Frontiers in Ecology and Evolution, 8, 1–12.
Beadleas, M.L., Gingrich, A.R. and Miller, J.A. (1977) Slow-Release devices for livestock insect
control: Cattle body surfaces contacted by five types of devices. Journal of Economic
Entomology, 70, 72–75.
Bedini, S., Flamini, G., Cosci, F., Ascrizzi, R., Echeverria, M.C., Gomez, E. V., Guidi, L., Landi, M.,
Lucchi, A. and Conti, B. (2019) Toxicity and oviposition deterrence of essential oils of
Clinopodium nubigenum and Lavandula angustifolia against the myiasis-inducing blowfly Lucilia
sericata. PLoS ONE, 14, 1–17.
Bedini, S., Guarino, S., Echeverria, M.C., Flamini, G., Ascrizzi, R., Loni, A. and Conti, B. (2020) Allium
sativum, rosmarinus officinalis, and salvia officinalis essential oils: A spiced shield against
blowflies. Insects, 11, 1–18.
Benelli, G. and Pavela, R. (2018a) Beyond mosquitoes—Essential oil toxicity and repellency against
bloodsucking insects. Industrial Crops and Products, 117, 382–392.
Benelli, G. and Pavela, R. (2018b) Repellence of essential oils and selected compounds against
ticks—A systematic review. Acta Tropica, 179, 47–54.
Bernier, U.R., Furman, K.D., Kline, D.L., Allan, S.A. and Barnard, D.R. (2005) Comparison of contact
and spatial repellency of catnip oil and N,N-Diethyl-3-methylbenzamide (DEET) against
mosquitoes. Journal of Medical Entomology, 42, 306–311.
Berry, I. L., & Kunz, S. E. (1977) Mortality of Adult Stable Flies. Environmental Entomology, 6, 569–
574.
Beynon, S.A., Wainwright, W.A. and Christie, M. (2015) The application of an ecosystem services
framework to estimate the economic value of dung beetles to the U.K. cattle industry.
Ecological Entomology, 40, 124–135.
Bischoff, K. and Guale, F. (1998) Australian tea tree (Melaleuca alternifolia) oil poisoning in three
purebred cats. Journal of Veterinary Diagnostic Investigation, 10, 208–210.
Blackwell, A., Stuart, A.E. and Estambale, B.A. (2003) The repellent and antifeedant activity of oil of
Myrica gale against Aedes aegypti mosquitoes and its enhancement by the addition of
48
salicyluric acid. The Journal of the Royal College of Physicians of Edinburgh, 33, 209-214.
Bohbot, J.D. and Dickens, J.C. (2010) Insect repellents: modulators of mosquito odorant receptor
activity. PLoS One, 5, 1-11
Bouzeraa, H., Bessila-Bouzeraa, M. and Labed, N., (2019) Repellent and fumigant toxic potential of
three essential oils against Ephestia kuehniella. Biosystems Diversity, 27, 349-353.
Braverman, Y., Ungar-Waron, H., Frith, K., Adler, H., Danieli, Y., Baker, K. and Quinn, P. (1983)
Epidemiological and immunological studies of sweet itch in horses in Israel. Veterinary Record,
112, 512–524.
Burgess, I.F. (2009) The mode of action of dimeticone 4% lotion against head lice, Pediculus capitis.
BioMed Central Pharmacology, 9, 1–8.
Burkhard, P.R., Burkhardt, K., Landis, T. and Haenggeli, C.A. (1999) Plant-induced seizures:
Reappearance of an old problem. Journal of Neurology, 246, 667–670.
Buschman, L.L. and Patterson, R.S. (1981) Assembly, mating, and thermoregulating behavior of
stable flies under field conditions. Environmental Entomology, 10, 16–21.
Butler, J.F., Kloft, W.J., DuBose, L.A. and Kloft, E.S. (1977) Recontamination of food after feeding a
32P food source to biting Muscidae. Journal of Medical Entomology, 13, 567-571.
Callander, J.T. and James, P.J. (2012) Insecticidal and repellent effects of tea tree (Melaleuca
alternifolia) oil against Lucilia cuprina. Veterinary Parasitology, 184, 271–278.
Campbell, J.B. (1977) Horn, face and stable fly control with feed additive insecticides. Insecticide and
Acaricide Tests, 2, 136–137.
Campbell, J.B., Skoda, S.R., Berkebile, D.R., Boxler, D.J., Thomas, G.D., Adams, D.C. and Davis, R.
(2001) Effects of stable flies (Diptera: Muscidae) on weight gains of grazing yearling cattle.
Journal of Economic Entomology, 94, 780–783.
Campbell, W.C. (1985) Ivermectin: An update. Parasitology Today, 1, 10–16.
Cantalamessa, F. (1993) Acute toxicity of two pyrethroids, permethrin, and cypermethrin in neonatal
and adult rats. Archives of Toxicology, 67, 510-513.
Cárdenas, J., Rojas, J., Rondón, M. and Nieves, E. (2012) Adulticide effect of Monticalia
greenmaniana (Asteraceae) against Lutzomyia migonei (Diptera: Psychodidae). Parasitology
Research, 111, 787–794.
Cavanagh, H.M.A. and Wilkinson, J.M. (2002) Biological activities of lavender essential
oil. Phytotherapy Research, 16, 301-308.
Centers for Disease Control and Prevention (2011) Guideline for Evaluating Insecticide Resistance in
Vectors Using the CDC Bottle Bioassay. Centers for Disease Control and Prevention, 11-14.
Chaaban, A., Gomes, E.N., Richardi, V.S., Martins, C.E.N., Brum, J.S., Navarro-Silva, M.A., Deschamps,
49
C. and Molento, M.B. (2019b) Essential oil from Curcuma longa leaves: Can an overlooked by-
product from turmeric industry be effective for myiasis control? Industrial Crops and Products,
132, 352–364.
Chaaban, A., Martins, C.E.N., Bretanha, L.C., Micke, G.A., Carrer, A.R., Rosa, N.F., Ferreira, L. and
Molento, M.B. (2018a) Insecticide activity of Baccharis dracunculifolia essential oil against
Cochliomyia macellaria (Diptera: Calliphoridae). Natural Product Research. 32, 2954–2958.
Chaaban, A., Richardi, V.S., Carrer, A.R., Brum, J.S., Cipriano, R.R., Martins, C.E.N., Silva, M.A.N.,
Deschamps, C. and Molento, M.B. (2019a) Insecticide activity of Curcuma longa (leaves)
essential oil and its major compound α-phellandrene against Lucilia cuprina larvae (Diptera:
Calliphoridae): Histological and ultrastructural biomarkers assessment. Pesticide Biochemistry
and Physiology, 153, 17–27.
Chaaban, A., Santos, V.M.C.S., Gomes, E.N., Martins, C.E.N., do Amaral, W., Deschamps, C. and
Molento, M.B. (2018b) Chemical composition of piper gaudichaudianum essential oil and its
bioactivity against Lucilia cuprina (Diptera: Calliphoridae). Journal of Essential Oil Research, 30,
159–166.
Chil-Núñez, I., Mendonça, P.M., Escalona-Arranz, J.C., Cortinhas, L.B., Dutok-Sánchez, C.M. and de
Carvalho Queiroz, M.M. (2018) Insecticidal effects of Ocimum sanctum var. cubensis essential
oil on the diseases vector Chrysomya putoria. Journal of Pharmacy and Pharmacognosy
Research, 6, 148–157.
Christen, V., Joho, Y., Vogel, M. and Fent, K. (2019) Transcriptional and physiological effects of the
pyrethroid deltamethrin and the organophosphate dimethoate in the brain of honeybees (Apis
mellifera). Environmental Pollution, 244, 247–256.
Cilek, J.E. (1999) Evaluation of various substances to increase adult Stomoxys calcitrans (Diptera:
Muscidae) collections on alsynite cylinder traps in North Florida. Journal of Medical
Entomology, 36, 605–609.
Cilek, J.E. and Greene, G.L. (1994) Stable fly (Diptera: Muscidae) insecticide resistance in Kansas
cattle feedlots. Journal of Economic Entomology, 87, 275–279.
Colwell, D.D., Kavaliers, M. and Lysk, T. (1997) Stable fly, Stomoxys calcitrans, mouthpart removal
influences stress and anticipatory responses in mice. Medical and Veterinary Entomology, 11,
310–314.Medical and Veterinary Entomology, 11, 310–314.
Corcos, D., Mazzon, L., Cerretti, P., Mei, M., Giussani, E., Drago, A. and Marini, L. (2019) Effects of
natural pyrethrum and synthetic pyrethroids on the tiger mosquito, Aedes albopictus (skuse)
and non-target flower-visiting insects in urban green areas of Padua, Italy. International Journal
of Pest Management, 66, 215–221.
50
Cossetin, L.F., Santi, E.M.T., Cossetin, J.F., Dillmann, J.B., Baldissera, M.D., Garlet, Q.I., De Souza, T.P.,
Loebens, L., Heinzmann, B.M., Machado, M.M. and Monteiro, S.G. (2018) In vitro safety and
efficacy of lavender essential oil (Lamiales: Lamiaceae) as an insecticide against houseflies
(Diptera: Muscidae) and blowflies (Diptera: Calliphoridae). Journal of Economic Entomology,
111, 1974–1982.
Costa-Júnior, L.M., Miller, R.J., Alves, P.B., Blank, A.F., Li, A.Y. and Pérez de León, A.A. (2016)
Acaricidal efficacies of Lippia gracilis essential oil and its phytochemicals against
organophosphate-resistant and susceptible strains of Rhipicephalus (Boophilus) microplus.
Veterinary Parasitology, 228, 60–64.
Costa, C.A.R. de A., Kohn, D.O., De Lima, V.M., Gargano, A.C., Flório, J.C. and Costa, M. (2011) The
GABAergic system contributes to the anxiolytic-like effect of essential oil from Cymbopogon
citratus (lemongrass). Journal of Ethnopharmacology, 137, 828–836.
Cox, S.D., Mann, C.M., Markham, J.L., Bell, H.C., Gustafson, J.E., Warmington, J.R. and Wyllie, S.G.
(2000) The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree
oil). Journal of Applied Microbiology, 88, 170-175.
Cox, S.D., Mann, C.M., Markham, J.L., Gustafson, J.E., Warmington, J.R. and Wyllie, S.G. (2001)
Determining the antimicrobial actions of tea tree oil. Molecules, 6, 87-91.
Cumming, J. (1998) Diptera associated with livestock dung. [online] North America Dipterists Society.
Available at: <http://www.nadsdiptera.org/FFP/stable.htm> [Accessed 9 April 2020].
Cutkomp, L.K. and Harvey, A.L. (1958) The weight responses of beef cattle in relation to control of
horn and stable flies. Journal of Economic Entomology, 51, 72–75.
de Oliveira, B.M.S., Melo, C.R., Alves, P.B., Santos, A.A., Santos, A.C.C., Santana, A.D.S., Araújo,
A.P.A., Nascimento, P.E.S., Blank, A.F. and Bacci, L. (2017) Essential oil of aristolochia trilobata:
Synthesis, routes of exposure, acute toxicity, binary mixtures and behavioral effects on leaf-
cutting ants. Molecules, 22, 1-17.
Dougherty, C.T., Knapp, F.W., Burrus, P.B., Willis, D.C. and Cornelius, P.L. (1994) Moderation of
grazing behavior of beef cattle by stable flies (Stomoxys calcitrans L.). Applied Animal Behaviour
Science, 40, 113–127.
Dougherty, C.T., Knapp, F.W., Burrus, P.B., Willis, D.C., Cornelius, P.L. and Bradley, N.W. (1993)
Multiple releases of stable flies (Stomoxys calcitrans L.) and behaviour of grazing beef
cattle. Applied Animal Behaviour Science, 38, 191-212.
Dsouli, N., Delsuc, F., Michaux, J., De Stordeur, E., Couloux, A., Veuille, M. and Duvallet, G. (2011)
Phylogenetic analyses of mitochondrial and nuclear data in haematophagous flies support the
paraphyly of the genus Stomoxys (Diptera: Muscidae). Infection, Genetics and Evolution, 11,
51
663-670.
El Ashmawy, W.R., Williams, D.R., Gerry, A.C., Champagne, J.D., Lehenbauer, T.W. and Aly, S.S.
(2019) Risk factors affecting dairy cattle protective grouping behavior, commonly known as
bunching, against Stomoxys calcitrans (L.) on California dairies. PLoS ONE, 14, 1–22.
Ellse, L. and Wall, R. (2014) The use of essential oils in veterinary ectoparasite control: A review.
Medical and Veterinary Entomology, 28, 233–243.
Ellse, L., Sands, B., Burden, F.A. and Wall, R. (2016) Essential oils in the management of the donkey
louse, Bovicola ocellatus. Equine Veterinary Journal, 48, 285–289.
Enan, E. (2001) Insecticidal activity of essential oils: Octopaminergic sites of action. Comparative
Biochemistry and Physiology - C Toxicology and Pharmacology, 130, 325–337.
Enan, E.E. (2005) Molecular and pharmacological analysis of an octopamine receptor from American
cockroach and fruit fly in response to plant essential oils. Archives of Insect Biochemistry and
Physiology, 59, 161–171.
Farhat, G.N., Affara, N.I. and Gali-Muhtasib, H.U. (2001) Seasonal changes in the composition of the
essential oil extract of East Mediterranean sage (Salvia libanotica) and its toxicity in mice.
Toxicon, 39, 1601–1605.
Farnsworth, W.R., Collett, M.G. and Ridley, I.S. (1997) Field Survey of Insecticide Resistance in
Haematobia irritans exigua de Meijere (Diptera: Muscidae). Australian Journal of Entomology,
36, 257–261.
Feaster, J.E., Scialdone, M.A., Todd, R.G., Gonzalez, Y.I., Foster, J.P. and Hallahan, D.L. (2009)
Dihydronepetalactones deter feeding activity by mosquitoes, stable flies, and deer ticks.
Journal of Medical Entomology, 46, 832–840.
Ferreira, T.P., Haddi, K., Corrêa, R.F.T., Zapata, V.L.B., Piau, T.B., Souza, L.F.N., Santos, S.M.G.,
Oliveira, E.E., Jumbo, L.O.V., Ribeiro, B.M., Grisolia, C.K., Fidelis, R.R., Maia, A.M.S. and Aguiar,
R.W.S. (2019) Prolonged mosquitocidal activity of Siparuna guianensis essential oil
encapsulated in chitosan nanoparticles. PLoS Neglected Tropical Diseases, 13, 1–23.
Florez-Cuadros, M., Berkebile, D., Brewer, G. and Taylor, D.B. (2019) Effects of diet quality and
temperature on stable fly (Diptera: Muscidae) development. Insects, 10, 1–13.
Foil, L.D. and Hogsette, J.A. (1994) Biology and control of tabanids, stable flies and horn flies. Revue
Scientifique et Technique, 13, 1125–1158.
Food and Agriculture Organization of the United Nations (2004) Food and Agriculture Organization
of the United Nations, Module 1. Ticks: acaricide resistance: diagnosis management and
prevention. In: Guidelines Resistance Management and Integrated Parasite Control In
Ruminants, FAO Animal Production and Health Division, Rome, Italy.
52
Fouche, G., Adenubi, O.T., Leboho, T., McGaw, L.J., Naidoo, V., Wellington, K.W. and Eloff, J.N.
(2019) Acaricidal activity of the aqueous and hydroethanolic extracts of 15 South African plants
against Rhipicephalus turanicus and their toxicity on human liver and kidney cells.
Onderstepoort Journal of Veterinary Research, 86, 1–7.
Fouche, G., Sakong, B.M., Adenubi, O.T., Dzoyem, J.P., Naidoo, V., Leboho, T., Wellington, K.W. and
Eloff, J.N. (2017) Investigation of the acaricidal activity of the acetone and ethanol extracts of
12 South African plants against the adult ticks of Rhipicephalus turanicus. Onderstepoort
Journal of Veterinary Research, 84, 1–6.
Galli, G.M., Roza, L.F., Santos, R.C., Quatrin, P.M., Ourique, A.F., Klein, B., Wagner, R., Baldissera,
M.D., Volpato, A., Campigotto, G. and Glombowsky, P. (2018) Low dose of nanocapsules
containing eucalyptus oil has beneficial repellent effect against horn fly (Diptera:
Muscidae). Journal of Economic Entomology, 111, 2983-2987.
Gebremichael, S., Birhanu, T. and Tessema, D.A. (2013) Analysis of organochlorine pesticide residues
in human and cow’s milk in the towns of Asendabo, Serbo and Jimma in South-Western
Ethiopia. Chemosphere, 90, 1652–1657.
George, D.R., Callaghan, K., Guy, J.H. and Sparagano, O.A. (2008) Lack of prolonged activity of
lavender essential oils as acaricides against the poultry red mite (Dermanyssus gallinae) under
laboratory conditions. Research in Veterinary Science, 85, 540–542.
Gilles, J., David, J.-F. and Duvallet, G. (2005) Temperature effects on development and survival of
two stable flies, Stomoxys calcitrans and Stomoxys niger niger (Diptera: Muscidae), in La
Réunion Island. Journal of Medical Entomology, 42, 260–265.
González, M., Venter, G.J., López, S., Iturrondobeitia, J.C. and Goldarazena, A. (2014) Laboratory and
field evaluations of chemical and plant-derived potential repellents against Culicoides biting
midges in northern Spain. Medical and Veterinary Entomology, 28, 421–431.
Goode, P., Ellse, L. and Wall, R. (2018) Preventing tick attachment to dogs using essential oils. Ticks
and Tick-borne Diseases, 9, 921-926.
Greive, K.A., Staton, J.., Miller, P.F., Peters, B.A. and Oppenheim, V.M.J. (2010) Development of
Melaleuca oils as effective natural-based personal insect repellents. Australian Journal of
Entomology, 49, 40–48.
Hagvall, L., Sköld, M., Bråred-Christensson, J., Börje, A. and Karlberg, A.T. (2008) Lavender oil lacks
natural protection against autoxidation, forming strong contact allergens on air exposure.
Contact Dermatitis, 59, 143–150.
Hammer, K., Carson, C.F. and Riley, T.V. (2003) Antifungal activity of the components of Melaleuca
alternifolia (tea tree) oil. Journal of Applied Microbiology, 95, 853-860.
53
Harris, R.L., Miller, J.A. and Frazar, E.D. (1974) Horn flies and stable flies: Feeding activity. Annals of
the Entomological Society of America, 67, 891-894.
Hazarika, S., Dhiman, S., Rabha, B., Bhola, R. and Singh, L. (2012) Repellent activity of some essential
oils against Simulium species in India. Journal of Insect Science, 12, 1–9.
Herath, H.M.W., Iruthayathas, E.E. and Orod, D.P. (1979) Temperature effects on essential oil
composition of citronella selections. Economic Botany, 33, 425–430.
Herholz, C., Kopp, C., Wenger, M., Mathis, A., Wägeli, S. and Roth, N. (2016) Efficacy of the repellent
N, N-diethyl-3-methyl-benzamide (DEET) against tabanid flies on horses evaluated in a field test
in Switzerland. Veterinary Parasitology, 221, 64-67.
Herman, A. and Herman, A.P. (2015) Essential oils and their constituents as skin penetration
enhancer for transdermal drug delivery: a review. Journal of Pharmacy and Pharmacology, 67,
473-485.
Hieu, T.T., Jung, J., Kim, S.I., Ahn, Y.-J. and Kwon, H.W. (2014) Behavioural and electroantennogram
responses of the stable fly (Stomoxys calcitrans L.) to plant essential oils and their mixtures
with attractants. Pest Management Science, 70, 163–172.
Hieu, T.T., Kim S.I., Kwon, H.W., and Ahn, Y.S. (2010a) Enhanced repellency of binary mixtures of
Zanthoxylum piperitum pericarp steam distillate or Zanthoxylum armatum seed oil constituents
and Calophyllum inophyllum nut oil and their aerosols to Stomoxys calcitrans. Pest
Management Science, 66, 1191–1198.
Hieu, T.T., Kim, S.I., Lee, S.-G. and Ahn, Y.J. (2010b) Repellency to Stomoxys calcitrans (Diptera:
Muscidae) of plant essential oils alone or in combination with Calophyllum inophyllum nut oil.
Journal of Medical Entomology, 47, 575–580.
Hogsette, J. A and J. P. Ruff. (1986) Evaluation of flucythrinate- and fenvalerate-impregnated ear
tags and permethrin ear tapes for fly control on beef and dairy cattle in northwest Florida.
Journal of Economic Entomology, 79, 152-157.
Hogsette, J.A. (1983) An attractant self-marking device for marking field populations of stable flies
with fluorescent dusts. Journal of Economic Entomology, 76, 510-514.
Hogsette, J.A. and Kline, D.L. (2017) The Knight Stick Trap and Knight Stick Sticky Wraps: New tools
for stable fly (Diptera: Muscidae) management. Journal of Economic Entomology, 110, 1384‐
1389
Hogsette, J.A. and Ose, G.A. (2017) Improved capture of stable flies (Diptera: Muscidae) by
placement of knight stick sticky fly traps protected by electric fence inside animal exhibit yards
at the Smithsonian’s National Zoological Park. Zoo Biology, 36, 382–386.
Hogsette, J.A. and Ruff, J.P. (1985) Stable fly (Diptera: Muscidae) migration in northwest
54
Florida. Environmental Entomology, 14, 170-175.
Hogsette, J.A., Ruff, J.P. and Jones, C.J. (1987) Stable fly biology and control in northwest Florida.
Journal of Agricultural Entomology, 4, 1–11.
Holdsworth, P.A., Vercruysse, J., Rehbein, S., Peter, R.J., De Bruin, C., Letonja, T. and Green, P. (2006)
World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines for
evaluating the efficacy of ectoparasiticides against biting and nuisance flies on
ruminants. Veterinary Parasitology, 136, 3-13.
Hollingworth, R.M., Ahammadsahib, K.I., Gadelhak, G. and McLaughlin, J.L. (1994) New inhibitors of
complex I of the mitochondrial electron transport chain with activity as pesticides. Biochemical
Society Transactions, 22, 230-233.
Holm, Y., Laakso, I., Hiltunen, R. and Galambosi, B. (1997) Variation in the essential oil composition
of Artemisia annua L. of different origin cultivated in Finland. Flavour and Fragrance Journal,
12, 241–246.
International Organization for Standardization (IOS), 2017. Essential oil of Melaleuca, terpinen-4-ol
type (tea tree oil). [online] Geneva: International Organization for Standardization. Available at:
<https://www.sis.se/api/document/preview/921469> [Accessed 22 March 2020].
Isman, M. B. (1997) Neem and other Botanical insecticides: Barriers to commercialisation.
Phytoparasitica, 25, 339.
Isman, M.B. (2006) Botanical insecticides, deterrents, and repellents in modern agriculture and an
increasingly regulated world. Annual Review Entomology, 51, 45–66.
Isman, M.B. (2017) Bridging the gap: moving botanical insecticides from the laboratory to the farm.
Industrial Crops and Products, 110, 10-14.
Isman, M.B. (2019) Commercial development of plant essential oils and their constituents as active
ingredients in bioinsecticides. Phytochemistry Reviews, 19, 235–241.
Isman, M.B. and Grieneisen, M.L. (2014) Botanical insecticide research: Many publications, limited
useful data. Trends in Plant Science, 19, 140–145.
James, P.J. and Callander, J.T. (2012) Dipping and jetting with tea tree (Melaleuca alternifolia) oil
formulations control lice (Bovicola ovis) on sheep. Veterinary Parasitology, 189, 338-343.
Jankowska, M., Rogalska, J., Wyszkowska, J. and Stankiewicz, M. (2018) Molecular targets for
components of essential oils in the insect nervous system—a review. Molecules, 23, 1–20.
Juan, L.W., Lucia, A., Zerba, E.N., Harrand, L., Marco, M. and Masuh, H.M. (2011) Chemical
composition and fumigant toxicity of the essential oils from 16 species of Eucalyptus against
Haematobia irritans (Diptera: Muscidae) adults. Journal of Economic Entomology, 104, 1087-
1092.
55
Karasek, I., Butler, C., Baynes, R. and Werners, A. (2020) A review on the treatment and control of
ectoparasite infestations in equids. Journal of Veterinary Pharmacology and Therapeutics, 0, 1-
8.
Khater, H.F. (2014) Bioactivities of some essential oils against the camel nasal botfly, Cephalopina
titillator. Parasitology Research, 113, 593–605.
Khater, H.F. and Geden, C.J. (2018) Potential of essential oils to prevent fly strike and their effects on
the longevity of adult Lucilia sericata. Journal of Vector Ecology, 43, 261–270.
Khater, H.F., Hanafy, A., Abdel-Mageed, A.D., Ramadan, M.Y. and El-Madawy, R.S. (2011) Control of
the myiasis-producing fly, Lucilia sericata, with Egyptian essential oils. International Journal of
Dermatology, 50, 187–194.
Khater, H.F., Ramadan, M.Y. and El-Madawy, R.S. (2009) Lousicidal, ovicidal and repellent efficacy of
some essential oils against lice and flies infesting water buffaloes in Egypt. Veterinary
Parasitology, 164, 257–266.
Killough, R. A., and Mckinstry, D. M. (1965) Mating and Oviposition Studies of the Stable Fly. Journal
of Economic Entomology, 58, 489–491.
Kim, S.I., Yoon, J.S., Baeck, S.J., Lee, S.H., Ahn, Y.J. and Kwon, H.W. (2012) Toxicity and synergic
repellency of plant essential oil mixtures with vanillin against Aedes aegypti (Diptera:
Culicidae). Journal of Medical Entomology, 49, 876-885.
Klauck, V., Pazinato, R., Radavelli, W.M., Volpato, A., Stefani, L.M. and da Silva, A.S. (2015) In vitro
repellent effect of tea tree (Melaleuca alternifolia) and andiroba (Carapa guianensis) oils on
Haematobia irritans and Chrysomya megacephala flies. Tropical Biomedicine, 32, 33-39.
Klauck, V., Pazinato, R., Stefani, L.M., Santos, R.C., Vaucher, R.A., Baldissera, M.D., Raffin, R., Boligon,
A., Athayde, M., Baretta, D. and Machado, G. (2014) Insecticidal and repellent effects of tea
tree and andiroba oils on flies associated with livestock. Medical and Veterinary
Entomology, 28, 33-39.
Kohari, D., Hongo, T. and Inoue, K. (2020) The influence of stable fly invasion on the behavior of
captive black rhinoceros (Diceros bicornis). Journal of Veterinary Behavior, 35, 83–87.
Kosgei, C.J., Matasyoh, J.C., Mwendia, C.M., Kariuki, S.T. and Guliye, A.Y. (2014) Chemical
composition and larvicidal activity of essential oil of Lippia kituiensis against larvae of
Rhipicephalus appendiculatus. International Journal of Biological and Chemical Sciences, 8,
1938–1947.
Koutsaviti, A., Antonopoulou, V., Vlassi, A., Antonatos, S., Michaelakis, A., Papachristos, D.P. and
Tzakou, O. (2018) Chemical composition and fumigant activity of essential oils from six plant
families against Sitophilus oryzae (Col: Curculionidae). Journal of Pest Science, 91, 873–886.
56
Lachance, S. and Grange, G. (2014) Repellent effectiveness of seven plant essential oils, sunflower oil
and natural insecticides against horn flies on pastured dairy cows and heifers. Medical and
Veterinary Entomology, 28, 193–200.
Lange, B.M., Mahmoud, S.S., Wildung, M.R., Turner, G.W., Davis, E.M., Lange, I., Baker, R.C.,
Boydston, R.A. and Croteau, R.B. (2011) Improving peppermint essential oil yield and
composition by metabolic engineering. Proceedings of the National Academy of Sciences, 108,
16944-16949
López, M.D. and Pascual-Villalobos, M.J. (2010) Mode of inhibition of acetylcholinesterase by
monoterpenoids and implications for pest control. Industrial Crops and Products, 31, 284–288.
López, M.D., Campoy, F.J., Pascual-Villalobos, M.J., Muñoz-Delgado, E. and Vidal, C.J. (2015)
Acetylcholinesterase activity of electric eel is increased or decreased by selected
monoterpenoids and phenylpropanoids in a concentration-dependent manner. Chemico-
Biological Interactions, 229, 36–43
Lwande, W., Ndakala, A.J., Hassanali, A., Moreka, L., Nyandat, E., Ndungu, M., Amiani, H., Gitu, P.M.,
Malonza, M.M. and Punyua, D.K. (1999) Gynandropsis gynandra essential oil and its
constituents as tick (Rhipicephalus appendiculatus) repellents. PhytoChemistry, 50, 401–405.
Lysk, T. (1993) Seasonal abundance of stable flies and house flies (Diptera:Muscidae) in dairies in
Alberta, Canada. Journal of Medical Entomology, 30, 888–895.
Machtinger, E.T., Leppla, N.C. and Hogsette, J.A. (2016) House and stable fly seasonal abundance,
larval development substrates, and natural parasitism on small equine farms in Florida.
Neotropical Entomology, 45, 433–440.
Maciel, M. V., Morais, S.M., Bevilaqua, C.M.L., Silva, R.A., Barros, R.S., Sousa, R.N., Sousa, L.C., Brito,
E.S. and Souza-Neto, M.A. (2010) Chemical composition of Eucalyptus spp. essential oils and
their insecticidal effects on Lutzomyia longipalpis. Veterinary Parasitology, 167, 1–7.
Maes, C., Bouquillon, S. and Fauconnier, M.L. (2019) Encapsulation of essential oils for the
development of biosourced pesticides with controlled release: A review. Molecules, 24, 1–17.
Mägi, E., Järvis, T. and Miller, I. (2006) Effects of different plant products against pig mange
mites. Acta Veterinaria Brno, 75, 283-287.
Mahmoud, S.S. and Croteau, R.B. (2001) Metabolic engineering of essential oil yield and composition
in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran
synthase. Proceedings of the National Academy of Sciences, 98, 8915-8920.
Markus Lange, B. and Turner, G.W. (2013) Terpenoid biosynthesis in trichomes—current status and
future opportunities. Plant Biotechnology Journal, 11, 2-22.
Masmeatathip, R., Ketavan, C. and Duvallet, G. (2006) Morphological studies of Stomoxys spp.
57
(Diptera: Muscidae) in central Thailand. Agriculture and Natural Resources, 40, 872-881.
Masotti, V., Juteau, F., Bessière, J.M. and Viano, J. (2003) Seasonal and phenological variations of the
essential oil from the narrow endemic species Artemisia molinieri and its biological activities.
Journal of Agricultural and Food Chemistry, 51, 7115–7121.
Mekonnen, A., Tesfaye, S., Christos, S.G., Dires, K., Zenebe, T., Zegeye, N., Shiferaw, Y. and Lulekal, E.
(2019) Evaluation of skin irritation and acute and subacute oral toxicity of Lavandula
angustifolia essential oils in rabbit and mice. Journal of Toxicology, 2019, 1-8.
Mellor, P.S., Kitching, R.P. and Wilkinson, P.J. (1987) Mechanical transmission of capripox virus and
African swine fever virus by Stomoxys calcitrans. Research in Veterinary Science, 43, 109–112.
Meyer, J.A. and Petersen, J.J. (1983) Characterization and seasonal distribution of breeding sites of
stable flies and house flies (Diptera: Muscidae) on eastern Nebraska feedlots and dairies.
Journal of Economic Entomology, 76, 103-108.
Miller, R.W., Pickens, L.G., Morgan, N.O., Thimlian, R.W. and Wilson, R.L. (1973) Effect of stable flies
on feed intake and milk production of dairy cows. Journal of Economic Entomology, 66, 711–
714.
Mills, C., Cleary, B. V., Walsh, J.J. and Gilmer, J.F. (2004) Inhibition of acetylcholinesterase by Tea
Tree oil. Journal of Pharmacy and Pharmacology, 56, 375–379.
Mills, P.C. (2007) Vehicle effects on the in vitro penetration of testosterone through equine
skin. Veterinary Research Communications, 31, 227-233.
Mills, P.C., Magnusson, B.M. and Cross, S.E. (2005) Effects of vehicle and region of application on
absorption of hydrocortisone through canine skin. American Journal of Veterinary Research, 66,
43-47.
Misharina, T.A., Polshkov, A.N., Ruchkina, E.L. and Medvedeva, I.B. (2003) Changes in the
composition of the essential oil in stored marjoram. Applied Biochemistry and Microbiology, 39,
353–358.
Mkolo, M.N. and Magano, S.R. (2007) Repellent effects of the essential oil of Lavendula angustifolia
against adults of Hyalomma marginatum rufipes. Journal of the South African Veterinary
Association, 78, 149–152.
Morgan, D., Bailie, H. (1980) A field trial to determine the effect of fly control using permethrin on
milk yields in dairy cattle in the UK. Veterinary Record, 106, 121-123.
Morrison, P.E., Venkatesh, K. and Thompson, B. (1982) The role of male accessory-gland substance
on female reproduction with some observations of spermatogenesis in the stable fly. Journal of
Insect Physiology, 28, 607–614.
Mossa, A.H., Mohafrash, S.M.M. and Chandrasekaran, N. (2018) Safety of natural insecticides: Toxic
58
effects on experimental animals. BioMed Research International, 2018, 1–17.
Mottet, R.S., Moon, R.D., Hathaway, M.R. and Martinson, K.L. (2018) Effectiveness of Stable Fly
Protectants on Adult Horses. Journal of Equine Veterinary Science, 69, 11–15.
Moyo, B. and Masika, P.J. (2013) Validation of the acaricidal properties of materials used in ethno-
veterinary control of cattle ticks. African Journal of Microbiology Research, 7, 4701–4706.
Mullens, B.A., Watson, D.W., Gerry, A.C., Sandelin, B.A., Soto, D., Rawls, D., Denning, S., Guisewite, L.
and Cammack, J. (2017) Field trials of fatty acids and geraniol applied to cattle for suppression
of horn flies, Haematobia irritans (Diptera: Muscidae), with observations on fly defensive
behaviors. Veterinary Parasitology, 245, 14–28.
Munoz-Bertomeu, J., Arrillaga, I., Ros, R. and Segura, J. (2006) Metabolic engineering of essential oil
yield in spike lavender. Plant Physiology, 142, 890-900.
Muraleedharan, K. (2005) Pyrethroid insecticides, Sumicidin and Butox for the effective control of
Stomoxys calcitrans. Journal of Parasitic Diseases, 29, 164–165.
NAF UK (2020) NAF Off Citronella. [online] Available at: <https://www.naf-equine.eu/uk/care/naf-
off-citronella> [Accessed 8 July 2020].
Naissance. 2020. Ethically sourced natural oils and ingredients, Naissance. [online] Available at:
<https://naissance.com/> [Accessed 18 April 2020].
Najafian, S. (2016) The effect of time and temperature on the shelf life of essential oils of Lavandula
officinalis. Journal of Essential Oil Research, 28, 413–420.
Nchu, F., Magano, S.R. and Eloff, J.N. (2012) In vitro anti-tick properties of the essential oil of Tagetes
minuta L. (Asteraceae) on Hyalomma rufipes (Acari: Ixodidae). Onderstepoort Journal of
Veterinary Research, 79, 1–5.
Ndungu, M., Lwande, W., Hassanali, A., Moreka, L. and Chhabra, S.C. (1995) Cleome monophylla
essential oil and its constituents as tick (Rhipicephalus appendiculatus) and maize weevil
(Sitophilus zeamais) repellents. Entomologia Experimentalis et Applicata, 76, 217–222.
Nerio, L.S., Olivero-Verbel, J. and Stashenko, E. (2010) Repellent activity of essential oils: A review.
Bioresource Technology, 101, 372–378.
Nieves, E., Méndez, J.F., Lias, J., Rondón, M. and Briceño, B. (2010) Actividad repelente de aceites
esenciales contra las picaduras de Lutzomyia migonei (Diptera: Psychodidae). Revista de
Biologia Tropical, 58, 1549–1560.
Odeniran, P.O., Macleod, E.T., Ademola, I.O. and Welburn, S.C. (2019) Molecular identification of
bloodmeal sources and trypanosomes in Glossina spp., Tabanus spp. and Stomoxys spp.
trapped on cattle farm settlements in southwest Nigeria. Medical and Veterinary Entomology,
33, 269–281.
59
Orchard, I. (1982) Octopamine in insects: neurotransmitter, neurohormone, and neuromodulator.
Canadian Journal of Zoology, 60, 659–669.
Ose, G.A. and Hogsette, J.A. (2014) Spatial distribution, seasonality and trap preference of stable fly,
Stomoxys calcitrans L. (Diptera: Muscidae), adults on a 12-hectare zoological park. Zoo Biology,
33, 228–233.
Oyedele, A. O., Gbolade, A. A., Sosan, M. B., Adewovin, F. B., Soyelu, O. L. and Orafidiva, O. O. (2002)
Formulation of an effective mosquito-repellent topical product from lemongrass oil.
Phytomedicine, 9, 259-262.
Papachristos, D.P., Karamanoli, K.I., Stamopoulos, D.C. and Menkissoglu‐Spiroudi, U. (2004) The
relationship between the chemical composition of three essential oils and their insecticidal
activity against Acanthoscelides obtectus (Say). Pest Management Science: formerly Pesticide
Science, 60, 514-520.
Park, C.G., Jang, M., Yoon, K.A. and Kim, J. (2016) Insecticidal and acetylcholinesterase inhibitory
activities of Lamiaceae plant essential oils and their major components against Drosophila
suzukii (Diptera: Drosophilidae). Industrial Crops and Products, 89, 507–513.
Park, I.K. (2014) Fumigant toxicity of oriental sweetgum (Liquidambar orientalis) and valerian
(Valeriana wallichii) essential oils and their components, including their acetylcholinesterase
inhibitory activity, against Japanese termites (Reticulitermes speratus). Molecules, 19, 12547–
12558.
Parravani, A., Chivers, C.A., Bell, N., Long, S., Burden, F. and Wall, R. (2019) Seasonal abundance of
the stable fly Stomoxys calcitrans in southwest England. Medical and Veterinary Entomology,
33, 485–490.
Patra, G., Behera, P., Das, S., Saikia, B., Ghosh, S., Biswas, P., Kumar, A., Alam, S., Kawlni, L.,
Lalnunpuia, C., Lalchhandama, C., Bachan, M. and Debbarma, A. (2018) Stomoxys calcitrans and
its importance in livestock : a review. International Journal of Advance Agricultural Research, 6,
30–37.
Pavela, R. (2014) Insecticidal properties of Pimpinella anisum essential oils against the Culex
quinquefasciatus and the non-target organism Daphnia magna. Journal of Asia-Pacific
Entomology, 17, 287–293.
Pavela, R. and Govindarajan, M. (2016) The essential oil from Zanthoxylum monophyllum a potential
mosquito larvicide with low toxicity to the non-target fish Gambusia affinis. Journal of Pest
Science, 90, 369–378.
Pavela, R. and Sedlák, P. (2018) Post-application temperature as a factor influencing the insecticidal
activity of essential oil from Thymus vulgaris. Industrial Crops and Products, 113, 46–49.
60
Pazinato, R., Klauck, V., Volpato, A., Tonin, A.A., Santos, R.C., de Souza, M.E., Vaucher, R.A., Raffin,
R., Gomes, P., Felippi, C.C. and Stefani, L.M. (2014) Influence of tea tree oil (Melaleuca
alternifolia) on the cattle tick Rhipicephalus microplus. Experimental and Applied Acarology, 63,
77-83.
Périno-Issartier, S., Ginies, C., Cravotto, G. and Chemat, F. (2013) A comparison of essential oils
obtained from lavandin via different extraction processes: Ultrasound, microwave,
turbohydrodistillation, steam and hydrodistillation. Journal of Chromatography A, 1305, 41-47.
Périno-Issartier, S., Ginies, C., Cravotto, G. and Chemat, F. (2013) A comparison of essential oils
obtained from lavandin via different extraction processes: Ultrasound, microwave,
turbohydrodistillation, steam and hydrodistillation. Journal of Chromatography A, 1305, 41-47.
Perry, N.B., Anderson, R.E., Brennan, N.J., Douglas, M.H., Heaney, A.J., McGimpsey, J.A. and
Smallfield, B.M. (1999) Essential oils from Dalmatian sage (Salvia officinalis L.): Variations
among individuals, plant parts, seasons, and sites. Journal of Agricultural and Food Chemistry,
47, 2048–2054.
Peterson, C.J., Nemetz, L.T., Jones, L.M. and Coats, J.R. (2002. Behavioral activity of catnip
(Lamiaceae) essential oil components to the German cockroach (Blattodea: Blattellidae).
Journal of Economic Entomology, 95, 377–380.
Pitzer, J.B., Kaufman, P.E. and Tenbroeck, S.H. (2010) Assessing permethrin resistance in the stable
fly (Diptera: Muscidae) in Florida by using laboratory selections and field evaluations. Journal of
Economic Entomology, 103, 2258–2263.
Pitzer, J.B., Kaufman, P.E., Geden, C.J. and Hogsette, J.A. (2011) The ability of selected pupal
parasitoids (Hymenoptera: Pteromalidae) to locate stable fly hosts in a soiled equine bedding
substrate. Environmental Entomology, 40, 88–93.
Pouokam, G.B., Album, W.L., Ndikontar, A.S. and Sidatt, M.E.H. (2017) A pilot study in cameroon to
understand safe uses of pesticides in agriculture, risk factors for farmers’ exposure and
management of accidental cases. Toxics, 5, 1–15.
Priestley, C.M., Williamson, E.M., Wafford, K.A. and Sattelle, D.B. (2003) Thymol, a constituent of
thyme essential oil, is a positive allosteric modulator of human GABA A receptors and a homo-
oligomeric GABA receptor from Drosophila melanogaster. British Journal of Pharmacology, 140,
1363–1372.
Rajkumar, V., Gunasekaran, C., Christy, I.K., Dharmaraj, J., Chinnaraj, P. and Paul, C.A. (2019) Toxicity,
antifeedant and biochemical efficacy of Mentha piperita L. essential oil and their major
constituents against stored grain pest. Pesticide Biochemistry and Physiology, 156, 138-144.
Rivaroli, D.C., Guerrero, A., Valero, M.V., Zawadzki, F., Eiras, C.E., del Mar Campo, M., Sañudo, C.,
61
Jorge, A.M. and do Prado, I.N. (2016) Effect of essential oils on meat and fat qualities of
crossbred young bulls finished in feedlots. Meat Science, 121, 278-284.
Rowshan, V., Bahmanzadegan, A. and Saharkhiz, M.J. (2013) Influence of storage conditions on the
essential oil composition of Thymus daenensis Celak. Industrial Crops and Products, 49, pp.97-
101.
Russell, M. (1999) ‘Toxicology of tea tree oil’, In I. Southwell and R. Lowe (ed.) Tea tree: the
genus Melaleuca. Amsterdam: Harwood Academic Publishers, 191-201.
Salem, A., Bouhsira, E., Liénard, E., Melou, A.B., Jacquiet, P. and Franc, M. (2012) Susceptibility of
two European strains of Stomoxys calcitrans (L.) to cypermethrin, deltamethrin, fenvalerate, λ-
cyhalothrin, permethrin and phoxim. International Journal of Applied Research in Veterinary
Medicine, 10, 249–257.
Sands, B., Ellse, L. and Wall, R. (2016) Residual and ovicidal efficacy of essential oil-based
formulations in vitro against the donkey chewing louse Bovicola ocellatus. Medical and
Veterinary Entomology, 30, 78–84.
Sands, B., Mgidiswa, N., Nyamukondiwa, C. and Wall, R. (2018) Environmental consequences of
deltamethrin residues in cattle feces in an African agricultural landscape. Ecology and
Evolution, 8, 2938–2946.
Savelev, S., Okello, E., Perry, N.S.L., Wilkins, R.M. and Perry, E.K. (2003) Synergistic and antagonistic
interactions of anticholinesterase terpenoids in Salvia lavandulaefolia essential oil.
Pharmacology Biochemistry and Behavior, 75, 661–668.
Schmidt, E., Bail, S., Buchbauer, G., Stoilova, I., Atanasova, T., Stoyanova, A., Krastanova, A. and
Jirovetz, L. (2009) Chemical composition, olfactory evaluation and antioxidant effects of
essential oil from Mentha x piperita. Natural Product Communications, 4, 1107–1112.
Schowalter, T. D. and Klowden M. J. (1979) Blood meal size of the stable fly, Stomoxys calcitrans,
measured by the HiCN method. Mosquito New, 39, 110-112.
Schwinghammer, K.A., Knapp, F.W. and Boling, J.A. (1987) Physiological and nutritional response of
beef steers to combined infestations of horn fly and stable fly (Diptera: Muscidae). Journal of
Economic Entomology, 80, 1294–1298.
Sfara, V., Zerba, E.N. and Alzogaray, R.A. 2009. Fumigant insecticidal activity and repellent effect of
five essential oils and seven monoterpenes on first-instar nymphs of Rhodnius prolixus. Journal
of Medical Entomology, 46, 511–515.
Shaw, A.O. and Atkeson, F.W. (1943) Effect of spraying cows with repellent type sprays as measured
by milk production. Journal of Dairy Science, 26, 179–187.
Shetta, A., Kegere, J. and Mamdouh, W. (2019) Comparative study of encapsulated peppermint and
62
green tea essential oils in chitosan nanoparticles: Encapsulation, thermal stability, in-vitro
release, antioxidant and antibacterial activities. International Journal of Biological
Macromolecules, 126, 731–742.
Showler, A.T. (2017) Botanically based repellent and insecticidal effects against horn flies and stable
flies (Diptera: Muscidae). Journal of Integrated Pest Management, 8, 1–11.
Showler, A.T. and Osbrink, W.L.A. (2015) Stable fly, Stomoxys calcitrans (L.), dispersal and governing
factors. International Journal of Insect Science, 7, 19–25.
Sienkiewicz, M., Głowacka, A., Kowalczyk, E., Wiktorowska-Owczarek, A., Jóźwiak-Bębenista, M. and
Łysakowska, M. (2014) The biological activities of cinnamon, geranium and lavender essential
oils. Molecules, 19, 20929-20940.
Sikkema, J., de Bont, J.A. and Poolman, B. (1995) Mechanisms of membrane toxicity of
hydrocarbons. Microbiological Reviews, 59, 201-222.
Silvestre, A.J.D., Cavaleiro, J.A.S., Delmond, B., Filliatre, C. and Bourgeois, G. (1997) Analysis of the
variation of the essential oil composition of Eucalyptus globulus Labill. from Portugal using
multivariate statistical analysis. Industrial Crops and Products, 6, 27–33.
Skovgård, H. (2004) Sustained releases of the pupal parasitoid Spalangia cameroni (Hymenoptera:
Pteromalidae) for control of house flies, Musca domestica and stable flies Stomoxys calcitrans
(Diptera: Muscidae) on dairy farms in Denmark. Biological Control, 30, 288–297.
Skovgård, H. and Nachman, G. (2012) Population dynamics of stable flies Stomoxys calcitrans
(Diptera: Muscidae) at an organic dairy farm in Denmark based on mark-recapture with
destructive sub-sampling. Environmental Entomology, 41, 20–29.
Suwannayod, S., Sukontason, K., Pitasawat, B., Junkum, A., Limspatham, K., Jones, M. and Somboon,
P. (2019) Synergistic toxicity of plant essential oils combined with pyrethroid insecticides
against blow flies and the house fly. Insects, 10, 1–16.
Swist, S.L., Wilkerson, M.J., Wyatt, C.R., Broce, A.B. and Kanost, M.R. (2002) Modulation of bovine
lymphocyte response by salivary gland extracts of the stable fly, Stomoxys calcitrans (Diptera:
Muscidae). Journal of Medical Entomology, 39, 900–907.
Talbert, R. and Wall, R. (2012) Toxicity of essential and non-essential oils against the chewing louse,
Bovicola (Werneckiella) ocellatus. Research in Veterinary Science, 93, 831–835.
Tawatsin, A., Thavara, U., Chansang, U., Chavalittumrong, P. and Boonruad, B. (2006) Field
evalutation of DEET, Repel Care, and three plant based essential oil repellents against
mosquitoes, black flies (Diptera: Simulidae) and land leeches (Arhynchobdellida) in Thailand.
Journal of American Mosquito Control Association, 22, 306–313.
Tawatsin, A., Wratten, S.D., Scott, R.R., Thavara, U., and Techadamrongsin, Y. (2001) Repellency of
63
volatile oils from plants against three mosquito vectors. Journal of Vector Ecology, 26, 76–82.
Taylor, D.B. and Berkebile, D. (2006) Comparative efficiency of six stable fly (Diptera: Muscidae)
traps. Journal of Economic Entomology, 99, 1415–1419.
Taylor, D.B., Friesen, K. and Zhu, J. (2017) Precipitation and temperature effects on stable fly
(Diptera: Muscidae) population dynamics. Environmental Entomology, 46, 434–439.
Taylor, D.B., Moon, R.D. and Mark, D.R. (2012) Economic Impact of Stable Flies (Diptera: Muscidae)
on Dairy and Beef Cattle Production. Journal of Medical Entomology, 49, 198–209.
Todd, D.H. (1964) The biting fly Stomoxys calcitrans (L.) in dairy herds in New Zealand. New Zealand
Journal of Agricultural Research, 7, 60–79.
Toledo, P.F.S., Ferreira, T.P., Bastos, I.M.A.S., Rezende, S.M., Viteri Jumbo, L.O., Didonet, J., Andrade,
B.S., Melo, T.S., Smagghe, G., Oliveira, E.E. and Aguiar, R.W.S. (2019) Essential oil from
Negramina (Siparuna guianensis) plants controls aphids without impairing survival and
predatory abilities of non-target ladybeetles. Environmental Pollution, 255, 1–12.
Tong, F. and Coats, J.R. (2010) Effects of monoterpenoid insecticides on [3H]-TBOB binding in house
fly GABA receptor and 36Cl- uptake in American cockroach ventral nerve cord. Pesticide
Biochemistry and Physiology, 98, 317–324.
Traversa, D., Otranto, D., Iorio, R., Carluccio, A., Contri, A., Paoletti, B., Bartolini, R. and Giangaspero,
A. (2008) Identification of the intermediate hosts of Habronema microstoma and Habronema
muscae under field conditions. Medical and Veterinary Entomology, 22, 283–287.
Turek, C. and Stintzing, F.C. (2013) Stability of essential oils: A review. Comprehensive Reviews in
Food Science and Food Safety, 12, 40–53.
Turell, M.J., Dohm, D.J., Geden, C.J., Hogsette, J.A. and Linthicum, K.J. (2010) Potential for stable flies
and house flies (Diptera: Muscidae) to transmit rift valley fever virus. Journal of the American
Mosquito Control Association, 26, 45-448.
Turell, M.J., Dohm, D.J., Geden, C.J., Hogsette, J.A. and Linthicum, K.J. (2010) Potential for stable flies
and house flies (Diptera: Muscidae) to transmit rift valley fever virus. Journal of the American
Mosquito Control Association, 26, 445–448.
Uddin, M.H., Shahjahan, M., Amin, A.R., Haque, M.M., Islam, M.A. and Azim, M.E. (2016) Impacts of
organophosphate pesticide, sumithion on water quality and benthic invertebrates in
aquaculture ponds. Aquaculture Reports, 3, 88-92.
Urban, J.E. and Broce, A. (1998) Flies and their bacterial loads in greyhound dog kennels in Kansas.
Current Microbiology, 36, 164–170.
Vetere, A., Bertocchi, M., Pelizzone, I., Moggia, E., Travaglino, C., Della Grotta, M., Casali, S., Gerosa,
S., Strada, L., Filia, K., Casalini, J., Parmigiani, E. and Di Ianni, F. (2020) Acute tea tree oil
64
intoxication in a pet cockatiel (Nymphicus hollandicus): A case report. BioMed Central
Veterinary Research, 16, 1–5.
Viljoen, A.M., Subramoney, S., Vuuren, S.F.V., Başer, K.H.C. and Demirci, B. (2005) The composition,
geographical variation and antimicrobial activity of Lippia javanica (Verbenaceae) leaf essential
oils. Journal of Ethnopharmacology, 96, 271–277.
Vitela-Mendoza, I., Cruz-Vázquez, C., Solano-Vergara, J. and Orihuela-Trujillo, A. (2016) Short
communication: Relationship between serum cortisol concentration and defensive behavioral
responses of dairy cows exposed to natural infestation by stable fly, Stomoxys calcitrans.
Journal of Dairy Science, 99, 9912–9916.
Wang, Q., Reddy, V.A., Panicker, D., Mao, H.Z., Kumar, N., Rajan, C., Venkatesh, P.N., Chua, N.H. and
Sarojam, R. (2016) Metabolic engineering of terpene biosynthesis in plants using a trichome‐
specific transcription factor Ms YABBY 5 from spearmint (Mentha spicata). Plant Biotechnology
Journal, 14, 1619-1632.
Wanzala, W., Hassanali, A., Mukabana, W.R. and Takken, W. (2014) Repellent activities of essential
oils of some plants used traditionally to control the brown ear tick, Rhipicephalus
appendiculatus. Journal of Parasitology Research, 2014, 1-10.
Wanzala, W., Takken, W., Mukabana, W.R., Pala, A.O. and Hassanali, A. (2012) Ethnoknowledge of
Bukusu community on livestock tick prevention and control in Bungoma district, western
Kenya. Journal of Ethnopharmacology, 140, 298–324.
Weaving, H., Sands, B. and Wall, R. (2020) Reproductive sublethal effects of macrocyclic lactones and
synthetic pyrethroids on the dung beetle Onthophagus similis. Bulletin of Entomological
Research, 110, 195-200.
Weinzierl, R.A. and Jones, C.J. (1998) Releases of Spalangia Nigroaenea and Muscidifurax Zaraptor
(Hymenoptera: Pteromalidae) increase rates of parasitism and total mortality of stable fly and
house fly (Diptera: Muscidae) pupae in Illinois cattle feedlots. Journal of Economic Entomology,
91, 1114–1121.
Wieman, G. A., Campbell, J. B., Deshazer, J. A., & Berry, I. L. (1992). Effects of stable flies (Diptera:
Muscidae) and heat stress on weight gain and feed efficiency of feeder Cattle. Journal of
Economic Entomology, 85, 1835–1842.
Williams, D.F. (1973) Sticky traps for sampling population of Stomoxys calcitrans. Journal of
Economic Entomology, 66, 1279–1280.
World Health Organisation (2009) Guidelines for Efficacy Testing of Mosquito Repellents for Human
Skin. Control of Neglected Tropical Diseases WHO Pesticide Evaluation Scheme. World Health
Organisation, 4-5.
65
World Health Organisation (2018) Test Procedures for Insecticide Resistance Monitoring in Malaria
Vector Mosquitoes. Global Malaria Programme. World Health Organisation, 9-19.
Yadav, E., Kumar, S., Mahant, S., Khatkar, S. and Rao, R. (2017) Tea tree oil: a promising essential oil.
Journal of Essential Oil Research, 29, 201–213.
Yaghoobi-Ershadi, M.R., Akhavan, A.A., Jahanifard, E., Vatandoost, H., Amin, G., Moosavi, L., Zahraei
Ramazani, A.R., Abdoli, H. and Arandian, M.H. (2006) Repellency effect of Myrtle essential oil
and DEET against Phlebotomus papatasi, under laboratory conditions. Iranian Journal of Public
Health, 35, 7–13.
Yeom, H.J., Jung, C.S., Kang, J., Kim, J., Lee, J.H., Kim, D.S., Kim, H.S., Park, P.S., Kang, K.S. and Park,
I.K. (2015) Insecticidal and acetylcholine esterase inhibition activity of asteraceae plant
essential oils and their constituents against adults of the German cockroach (Blattella
germanica). Journal of Agricultural and Food Chemistry, 63, 2241–2248.
Yeruham, I. and Braverman, Y. (1995) Skin lesions in dogs, horses and calves caused by the stable fly
Stomoxys calcitrans (L.) (Diptera: Muscidae). Revue d’élevage et de Médecine Vétérinaire des
Pays Tropicaux, 48, 347–349.
Zeil, J. (1983) Sexual dimorphism in the visual system of flies: The free flight behaviour of male
Bibionidae (Diptera). Journal of Comparative Physiology, 150, 395–412.
Zhu, J.J., Berkebile, D.R., Dunlap, C.., Zhang, A., Boxler, D., Tangtrakulwanich, K., Behle, R..,
Baxendale, F. and Brewer, G. (2012) Nepetalactones from essential oil of Nepeta cataria
represent a stable fly feeding and oviposition repellent. Medical and Veterinary Entomology,
26, 131–138.
Zhu, J.J., Dunlap, C.A., Behle, R.W., Berkebile, D.R. and Wienhold, B. (2010) Repellency of a wax-
based catnip-oil formulation against stable flies. Journal of agricultural and food chemistry, 58,
12320-12326.
Zhu, J.J., Li, A.Y., Pritchard, S., Tangtrakulwanich, K., Baxendale, F. and Brewer, G. (2011) Contact and
fumigant toxicity of a botanical-based feeding deterrent of the stable fly, Stomoxys calcitrans
(Diptera: Muscidae). Journal of Agricultural and Food Chemistry, 59, 10394–10400.
Zhu, J.J., Wienhold, B.J., Wehrle, J., Davis, D., Chen, H., Taylor, D., Friesen, K. and Zurek, L. (2014)
Efficacy and longevity of newly developed catnip oil microcapsules against stable fly oviposition
and larval growth. Medical and Veterinary Entomology, 28, 222–227.
Zhu, J.J., Zeng, X.P., Berkebile, D., Du, H.J., Tong, Y. and Qian, K. (2009) Efficacy and safety of catnip
(Nepeta cataria) as a novel filth fly repellent. Medical and Veterinary Entomology, 23, 209–216.
Zumpt, F. (1973) The stomoxyine biting flies of the world. Stuttgart: G. Fischer.
66
Appendix
Ap
pen
dix I. A
n en
um
eration
of essen
tial oils w
hich
have b
een in
vestigated
for th
eir repellen
t or in
secticidal p
rop
erties again
st bitin
g flies of veterin
ary im
po
rtance.
Plan
t Family
Plan
t Species
(Co
mm
on
nam
e) Fly Sp
ecies (C
om
mo
n n
ame)
Bio
assay R
esults
Referen
ce
Am
aryllidace
ae
Alliu
m cep
a
(On
ion
) V
ariety IV
2.9 m
L/kg of b
uffalo
bo
dy w
eight d
eterred flies fo
r 6 days.
Kh
ater et al., 200
9
Alliu
m sa
tivum
(G
arlic) C
eph
alo
pina
titillato
r (C
amel n
asal bo
tfly)
LIB
LD50 w
as 0.44% (v/v).
Kh
ater, 2014.
Ca
lliph
ora vo
mito
ria
(blu
e bo
ttle blo
wfly)
TA
LD50 w
as 22% (v/v).
B
edin
i et al., 202
0
Ap
iaceae
C
orian
dru
m sa
tivum
(C
orian
der)
Stom
oxys ca
lcitrans
(Stable fly)
SB
0.5 mg/cm
2 repelled
flies for 12 m
inu
tes.
Hieu
et al., 201
0
Levisticum
officin
ale
(Lovage)
Stom
oxys ca
lcitrans
(Stable fly)
SB
0.5 mg/cm
2 repelled
flies for 3.36 h
ou
rs.
Hieu
et al., 201
0
Pim
pin
ella a
nisu
m
(An
ise)
Lucilia
sericata
(C
om
mo
n green
bo
ttle fly)
IB
LD50 fo
r larvae was 2.74
% (v/v).
Kh
ater et al., 201
1
Ara
ceae
H
om
alo
mena
aro
ma
tica
Scho
tt
Simu
lium
spp
. (B
lackflies)
SB
5% (v/v) rep
elled
flies for 2.13 h
ou
rs. H
azarika et al.,
2012
Asterace
ae
Ag
eratu
m con
zoid
es (B
illygoat-w
eed
)
Simu
lium
spp
. (B
lackflies)
SB
5% (v/v) rep
elled
flies for 2.85 h
ou
rs. H
azarika et al.,
2012
Artem
esia vu
lgaris
(Arm
oise)
Stom
oxys ca
lcitrans
(Stable fly)
SB
0.5 mg/cm
2 of rep
elled
flies for 20 m
inu
tes.
Hieu
et al., 201
0
Ba
ccharis d
racu
nculifo
lia
C
och
liomyia
ma
cellaria
(Secon
dary scre
ww
orm
) FP
B
LD50 fo
r larvae was 2.63μ
L/cm2.
Ch
aaban
et al.,
2018
Espeletia
shu
ltzii
Lutzo
myia
migo
nei
SB
0.416 μ
L/cm2 rep
elled flies fo
r 32 m
inu
tes. N
ieves et al., 2010
Lactu
ca sa
tiva
(Lettu
ce)
Lucilia
sericata
(C
om
mo
n green
bo
ttle fly)
IB
LD50 w
as 0.57% (v/v).
K
hater et a
l., 2011
Ma
tricaria
cham
omilla
(C
ham
om
ile) V
ariety IV
3.4 m
L/kg of b
uffalo
bo
dy w
eight d
eterred flies fro
m
bu
ffaloes fo
r 6 days.
Kh
ater et al., 2009
Lucilia
sericata
(C
om
mo
n green
bo
ttle fly)
IB
LD50 w
as 0.85% (v/v).
K
hater et a
l., 2011
Mo
ntica
lia
green
ma
nian
a
Lutzo
myia
migo
nei
FP
B
0.1 mg/m
L caused
100%
mo
rtality 1 ho
ur p
ost exp
osu
re
Card
enas et a
l., 2012
Mo
ntica
lia im
brica
tifolia
Lutzo
myia
migo
nei
SB
0.416 μ
L/cm2 rep
elled flies fo
r 1.45 ho
urs.
Nieves et a
l., 2010
Pseud
ogn
apha
lium
ca
eruleo
canum
Lutzo
myia
migo
nei
SB
0.416 μ
L/cm2 rep
elled flies fo
r 5 ho
urs.
Nieves et a
l., 2010
Cu
curb
itaceae
C
ucurb
ita m
axim
a
(Pu
mp
kin)
Cep
ha
lop
ina titilla
tor
(Cam
el nasal b
otfly)
LIB
LD50 w
as 0.20% (v/v).
Kh
ater, 2014.
Geran
iaceae
P
elargo
nium
gra
veolen
s (G
eraniu
m)
Stom
oxys ca
lcitrans
(Stable fly)
SB
0.5 mg/cm
2 repelled
flies for 1.11 h
ou
rs.
Hieu
et al., 201
0
Variety
IV
5% (v/v) sign
ificantly red
uced
the ab
un
dan
ce o
f flies on
h
eifers for 3 h
ou
rs.
Lachan
ce and
G
range
2014
Fabace
ae
Lup
inus lu
teus (Yello
w lu
pin
) C
eph
alo
pina
titillato
r (C
amel n
asal bo
tfly)
LIB
LD50 w
as 0.42% (v/v).
Kh
ater, 2014.
Lamiace
ae
Clin
opo
dium
nub
igenu
m
(Ku
nth
)
Lucilia
sericata
(C
om
mo
n green
bo
ttle fly)
FPB
LD50 fo
r eggs and
adu
lts was 0.07
μL/cm
2 and
0.278 μ
L/cm2,
respectively.
Bed
ini et a
l., 2019
Hyp
tis suaveo
lens (P
ignu
t) Lu
tzom
yia m
igon
ei
SB
No
repellen
t effect. N
ieves et al., 2010
Lavan
dula
ang
ustifolia
(En
glish Laven
der)
Lucilia
sericata
(C
om
mo
n green
bo
ttle fly)
BB
LD
50 was 0.063
% (v/v), 5 m
inu
tes po
st expo
sure.
Kh
ater and
Ged
en
2018
Lucilia
sericata
(C
om
mo
n green
bo
ttle fly)
FPB
LD50 fo
r eggs and
adu
lts was 0.48 μ
L/cm2 an
d 0.393 μ
L/ cm2,
respectively.
Bed
ini et a
l., 2019
Variety
IV
5% (v/v) sign
ificantly red
uced
the ab
un
dan
ce o
f flies on
h
eifers for 3 h
ou
rs.
Lachan
ce and
G
range
2014
Cu
licoid
es obso
letus
DC
B
1 μg/μ
L repelled
93.7%
of flies fo
r 4 min
utes.
G
on
zalez et al.,
2014
Stom
oxys ca
lcitrans
(Stable fly)
SB
0.5 mg/cm
2 repelled
flies for 29 m
inu
tes.
Hieu
et al., 201
0
Lavan
dula
den
tata
(Fren
ch Laven
der)
Ch
rysom
ya a
lbicep
s W
iedem
ann
FP
B
LD50 fo
r adu
lts was 5.14%
(lw/v).
Co
ssetin et a
l., 2018
Melissa
officina
lis (Le
mo
n b
alm)
Cu
licoid
es obso
letus
DC
B
1 μg/μ
L repelled
88.4%
of flies fo
r 4 min
utes.
G
on
zalez et al.,
2014
Men
tha
pip
erita
(Pep
pe
rmin
t) C
eph
alo
pina
titillato
r (C
amel n
asal bo
tfly)
LIB
LD50 w
as 0.47% (v/v).
Kh
ater, 2014.
Variety
In vivo
3.6 m
L/kg of b
uffalo
bo
dy w
eight d
eterred flies fo
r 6 days.
K
hater et al., 2
009
Variety
IV
5% (v/v) sign
ificantly red
uced
the ab
un
dan
ce o
f flies on
h
eifers for 3 h
ou
rs.
Lachan
ce and
G
range
2014
Plectra
nthu
s ambo
inicu
s
Lutzo
myia
migo
nei
SB
0.416 μ
L/cm2 rep
elled flies fo
r 4.18 ho
urs
Nieves et a
l., 2010
Nep
eta ca
taria
(Catn
ip)
Stom
oxys ca
lcitrans
(Stable fly)
NC
B
66 μg/μ
L repelled
97%
of flies fo
r 4 ho
urs.
Zhu
et al., 2009.
Stom
oxys ca
lcitrans
(Stable fly)
TA
FT
50 μg/fly cau
sed 1
00% m
ortality w
hen
top
ically ap
plied
and
th
e fum
igant LD
50 was 10.7 m
g/cm3.
Zhu
et al., 2011
Stom
oxys ca
lcitrans
(Stable fly)
NC
B
IV
67 μg/μ
L repe
lled 9
6% fro
m fee
din
g for 4 h
ou
rs. In vivo
, 15%
(v/v) EO rep
elled
flies for 6 h
ou
rs.
Zhu
et al., 2012
Ha
ema
tobia
irritans
(Ho
rn fly)
NC
B
0.67 μg/μ
L in h
exan
e, rep
elled 8
5% o
f flies for 4 h
ou
rs. Zh
u et a
l., 2015
Ocim
um
ba
silicum
(B
asil) V
ariety IV
5%
(v/v) significan
tly redu
ced th
e abu
nd
ance
of flies o
n
heifers fo
r 3 ho
urs.
Lachan
ce and
G
range
2014
Ocim
um
gra
tissimum
Lucilia
cup
rina
(A
ustralian
shee
p
blo
wfly)
Ch
rysom
ya m
egacep
ha
la
(Orien
tal latrine fly)
Ch
rysom
ya ru
fifacies (H
airy maggo
t blo
wfly)
TA
LD50 fo
radu
lts was 11
0, 166
and
68.5 μ
g/fly for th
e three
species, resp
ectively.
Suw
ann
ayod
et al.,
2019
Ocim
um
sanctu
m va
r. cu
ben
sis (H
oly b
asil)
Ch
rysom
ya pu
toria
(A
frican latrin
e blo
wfly)
TA
LD50 fo
r larvae was 7.47 m
g/mL.
Ch
il-Nu
nez et a
l., 2018.
Orig
anu
m m
ajo
rana
(M
arjoram
) Sto
mo
xys calcitran
s (Stab
le fly) SB
0.5 m
g/cm2 rep
elled flies fo
r 7 min
utes.
H
ieu et a
l., 2010
Orig
anu
m vu
lgare
(Oregan
o)
Stom
oxys ca
lcitrans
(Stable fly)
SB
0.5 mg/cm
2 repelled
flies for 1.15 h
ou
rs.
Hieu
et al., 201
0
Po
gostem
on ca
blin
(B
lanco
) (P
atcho
uli)
Stom
oxys ca
lcitrans
(Stable fly)
SB
0.5 mg/cm
2 repelled
flies for 3.67 h
ou
rs.
Hieu
et al., 201
0
Po
gostem
on h
eynean
us
Sim
uliu
m sp
p.
(Blackflies)
IV
SB
5% (v/v) p
rovid
ed p
rotectio
n fo
r 1.11 ho
urs.
Hazarika et a
l., 2012
Ro
sma
rinu
s officin
alis
(Ro
semary)
Stom
oxys ca
lcitrans
(Stable fly)
SB
0.5 mg/cm
2 repelled
flies for 13 m
inu
tes.
Hieu
et al., 201
0
Lu
cilia serica
ta
(Co
mm
on
green b
ottle
fly)
IB
LD50 w
as 6.77%
(v/v).
Kh
ater et al., 201
1
Cu
licoid
es obso
letus
DC
B
1 μg/μ
L repelled
70%
of flies fo
r 4 min
utes.
G
on
zalez et al.,
2014
Ca
lliph
ora vo
mito
ria
(blu
e bo
ttle blo
wfly)
TA
LD50 w
as 55% (v/v).
B
edin
i et al., 202
0
Salvia
officin
alis
(com
mo
n sage)
Ca
lliph
ora vo
mito
ria
(blu
e bo
ttle blo
wfly)
TA
LD50 w
as 99% (v/v).
B
edin
i et al., 202
0
Salvia
sclerea
(Sage) Sto
mo
xys calcitran
s (Stab
le fly)
SB
0.5 mg/cm
2 repelled
flies for 30 m
inu
tes.
Hieu
et al., 201
0
Satu
reja m
ona
ta
(Savory)
Stom
oxys ca
lcitrans
(Stable fly)
SB
0.5 mg/cm
2 did
no
t repe
l flies.
Hieu
et al., 201
0
Thym
us vu
lgaris
(Thym
e) Sto
mo
xys calcitran
s (Stab
le fly)
SB
0.5 mg/cm
2 repelled
flies for 2.12 h
ou
rs.
Hieu
et al., 201
0
Vitex n
egu
ndo
Simu
lium
spp
. (B
lackflies)
IV
SB
5% (v/v) EO
pro
vided
pro
tection
for 2.68 h
ou
rs H
azarika et al.,
2012
Lauraceae
C
inn
amo
mu
m ca
mp
hora
(C
amp
ho
r) V
ariety IV
1.4 m
L/kg of b
uffalo
bo
dy w
eight d
eterred flies fo
r 6 days
K
hater et al., 2
009
Cin
nam
om
um
verum
(Tru
e cinn
amo
n tree)
Lucilia
sericata
(C
om
mo
n green
bo
ttle fly)
BB
LD
50 for ad
ults w
as 0.079%
(v/v), 5 min
utes p
ost exp
osu
re. K
hater an
d G
ede
n
2018
Lu
tzom
yia m
igon
ei
SB
100% EO
pro
vided
pro
tection
for 4.2 h
ou
rs N
ieves et al., 2010
Meliace
ae
Ca
rapa
gu
ianen
sis (A
nd
irob
a) H
aem
atob
ia irritan
s (L.) (H
orn
fly) TA
IV
1%
(v/v) caused
100%
mo
rtality 4 ho
urs p
ost treatm
ent. 5%
(v/v) sign
ificantly red
uced
flies on
cattle for 6 h
ou
rs. K
lauck et a
l., 2014
Ha
ema
tobia
irritans (L.)
(Ho
rn fly)
FR
5% (v/v) rep
elled
flies for 3 h
ou
rs. K
lauck et a
l., 2015
Myrtace
ae
Co
rymb
ia citriod
ora
(Lem
on
-scen
ted gu
m)
Lutzo
myia
long
ipa
lpis
B
B
10% (v/v) ach
ieved 88.13%
mo
rtality 24 ho
urs p
ost
treatmen
t. M
aciel et al., 2
010
Cu
licoid
es obso
letus
DC
B
1 μg/μ
L repelled
90.5%
of flies.
G
on
zalez et al.,
2014
Euca
lyptus g
lobu
les (Eu
calyptu
s) Sto
mo
xys calcitran
s (Stab
le fly) SB
0.5 m
g/cm2 rep
elled flies fo
r 8 min
utes.
H
ieu et a
l., 2010
Lutzo
myia
long
ipa
lpis
B
B
10% (v/v) ach
ieved 95.50%
mo
rtality 24 ho
urs p
ost
treatmen
t.
Maciel et a
l., 2010
Euca
lyptus sta
igeria
na
(Lem
on
-scen
ted
iron
bark)
Lutzo
myia
long
ipa
lpis
B
B
5% (v/v) ach
ieved 9
9.62% m
ortality 24 h
ou
rs po
st treatmen
t. M
aciel et al., 2
010
Eug
enia
caryo
phylla
ta
(Clo
ve)
Stom
oxys ca
lcitrans
(Stable fly)
SB
0.5 mg/cm
2 repelled
flies for 3.5 h
ou
rs.
Hieu
et al., 201
0
Mela
leuca
altern
ifolia
(Tea tree)
Ha
ema
tobia
irritans (L.)
(Ho
rn fly)
TA
IV
1% (v/v) EO
caused
100%
mo
rtality 4 ho
urs p
ost treatm
ent.
5% (v/v) EO
significan
tly redu
ced flies o
n cattle fo
r 24 ho
urs.
Klau
ck et al., 20
14
Lucilia
cup
rina
(A
ustralian
shee
p
blo
wfly)
DC
B
FPB
IB
1% (v/v) fo
rmu
lation
caused
100%
ovicid
al and
larvicidal
mo
rtality. 3% (v/v) so
lutio
n p
revented
ovip
ositio
n o
f gravid
females fo
r 6 wee
ks.
Callan
der an
d
James 2012
.
Ha
ema
tobia
irritans (L.)
(Ho
rn fly)
FR
5% (v/v) rep
elled
flies for 2 h
ou
rs. K
lauck et a
l., 2015
Variety
IV
5% (v/v) sign
ificantly red
uced
the ab
un
dan
ce o
f flies on
h
eifers for 8 h
ou
rs. Lach
ance an
d
Gran
ge 2014
Myrtu
s comm
un
is (M
yrtle)
Ph
lebo
tomus pa
pa
tasi
NC
B
ED50 to
repel flies w
as for 5 m
inu
tes was 0.114 m
g/cm2
Yagh
oo
bi-Ersh
adi
et al., 200
6
Pim
enta
racem
ose
(West In
dian
bay tree)
Lutzo
myia
migo
nei
SB
N
o rep
ellent effect
Nieves et a
l., 2010
Psid
ium g
uaja
va
(Co
mm
on
guava)
Simu
lium
spp
. (B
lackflies)
IV
SB
10% (w
/w) EO
form
ulatio
n p
rovid
ed 1
00%
pro
tection
for 9
ho
urs.
Tawatsin
et al.,
2006
Oleaceae
Jasm
inum
gra
nd
iflorum
(Jasm
ine)
Cu
licoid
es obso
letus
DC
B
1 μg/μ
L repelled
93.9%
of flies.
G
on
zalez et al.,
2014
Pin
aceae
P
inu
s sylvestris (P
ine)
Variety
IV
5% (v/v) EO
caused
significan
tly low
er nu
mb
er of flies o
n
ind
ividu
al heifers 2 h
ou
rs po
st treatmen
t.
Lachan
ce and
G
range
2014
P
iperace
ae
Pip
er gau
dicha
ud
ianu
m
(Pip
er) Lu
cilia cu
prin
a
(Au
stralian sh
eep
b
low
fly)
FPB
LD
50 against larvae w
as 2.19 μ
L/cm2, 48 h
ou
rs po
st expo
sure.
Ch
aaban
et al.,
2018
Pip
er ma
rgin
atu
m
(Marigo
ld p
epp
er)
Lutzo
myia
migo
nei
SB
N
o rep
ellent effect.
Nieves et a
l., 2010
Po
aceae
Ch
rysopo
gon
zizan
ioid
es (V
etiver)
Lucilia
sericata
(C
om
mo
n green
bo
ttle fly)
BB
LD
50 for ad
ults w
as 0.082% (v/v), 5 m
inu
tes po
st expo
sure.
Kh
ater and
Ged
en
2018
Cym
bop
ogon
citratus
(Citro
nella)
Stom
oxys ca
lcitrans
(Stable fly)
DC
B
0.1 mg/μ
L EO rep
elled
flies from
feed
ing fo
r 10 min
utes.
B
aldacch
ino
et al.,
2013
Variety
IV
5% (v/v) EO
significan
tly redu
ced th
e abu
nd
ance
of flies o
n
heifers fo
r 3 ho
urs.
Lachan
ce and
G
range
2014
Cu
licoid
es obso
letus
DC
B
1 μg/μ
L repelled
72.7%
of flies.
G
on
zalez et al.,
2014
Stom
oxys ca
lcitrans
(Stable fly)
IV
6% (v/v) EO
form
ulatio
n sign
ificantly red
uced
fly ann
oyan
ce
beh
aviou
rs in h
orses fo
r 2 ho
urs.
Mo
ttet et al., 20
18
Cym
bop
ogon
nardu
s (C
itron
ella)
Stom
oxys ca
lcitrans
(Stable fly)
SB
0.5 mg/cm
2 repelled
flies for 16 m
inu
tes.
Hieu
et al., 201
0
Ha
ema
tobia
irritans (L.)
(Ho
rn fly)
FR
5% (v/v) EO
dilu
ted w
ith trito
n-w
ater solu
tion
repelled
flies fo
r 2 ho
urs.
Klau
ck et al., 20
15
Ru
taceae
A
myris ba
lsamifera
(W
est Ind
ian
sand
alwo
od
)
Stom
oxys ca
lcitrans
(Stable fly)
NC
B
67 μ
g/μL rep
elled 5
5% o
f flies from
feed
ing fo
r 4 ho
urs.
Zhu
et al., 2012
Citru
s auran
tifolia
Sw
ing
le
Simu
lium
spp
. (B
lackflies)
IV
SB
5% (v/v) EO
pro
vided
pro
tection
for 52 m
inu
tes. H
azarika et al.,
2012
Citru
s berg
am
ia (R
isso)
(Bergam
ot)
Stom
oxys ca
lcitrans
(Stable fly)
SB
0.5 mg/cm
2 repelled
flies for 37 m
inu
tes.
Hieu
et al., 201
0
Zan
thoxylu
m arm
atu
m
(Xan
tho
xylum
)
Stom
oxys ca
lcitrans
(Stable fly)
SB
0.5 mg/cm
2 repelled
flies for 35 m
inu
tes.
Hieu
et al., 201
0
Sto
mo
xys calcitran
s (Stab
le fly) SB
0.20 m
g/cm2 rep
elled 9
1% o
f flies for 3
0 min
utes.
H
ieu et a
l., 2010a
Sto
mo
xys calcitran
s (Stab
le fly) D
CB
0.06 m
g/μL rep
elled
86%
of flies fo
r 15 m
inu
tes.
Hieu
et al., 201
4
Za
ntho
xylum
pip
eritum
(Jap
anese p
epp
er) Sto
mo
xys calcitran
s (Stab
le fly) D
CB
0.06 m
g/μL rep
elled
87%
of flies fo
r 15 m
inu
tes.
Hieu
et al., 201
4
Sto
mo
xys calcitran
s (Stab
le fly) SB
0.4 m
g/cm2 rep
elled 7
2%
of flies fo
r 1.5 ho
urs.
Hieu
et al., 201
0a
Santalace
ae
San
talu
m a
lbum
(San
dalw
oo
d)
Stom
oxys ca
lcitrans
(Stable fly)
SB
0.5 mg/cm
2 repelled
flies for 16 m
inu
tes.
Hieu
et al., 201
0
Stom
oxys ca
lcitrans
(Stable fly)
NC
B
67 μ
g/μL rep
elled 7
0% o
f flies from
feed
ing fo
r 4 ho
urs.
Zhu
et al., 2012
Zingib
eraceae
B
oesenb
ergia
rotun
da
(Fingerro
ot)
Simu
lium
spp
. (B
lackflies)
IV
SB
10% (w
/w) p
rovid
ed 1
00% p
rotectio
n fo
r 9 ho
urs.
Tawatsin
et al.,
2006
Bio
assays used
for assessm
ent o
f essential o
ils: LIB, Larval Im
mersio
n B
ioassay; TA
, Top
ical Ap
plicatio
n; IB
, Ingestio
n B
ioassay; B
B, B
ottle B
ioassay; FP
B,
Filter Pap
er Bio
assay; FT, Fum
igant To
xicity; DC
B, D
ual C
ho
ice Bio
assay; NC
B, N
o C
ho
ice Bio
assay; SB, Skin
Bio
assay; IV, In
Vivo
.
Lucilia
cup
rina
(A
ustralian
shee
p
blo
wfly)
Ch
rysom
ya m
egacep
ha
la
(Orien
tal latrine fly)
Ch
rysom
ya ru
fifacies (H
airy maggo
t blo
wfly)
TA
LD50 again
st adu
lts was 207, 2
50 and
104 μ
g/fly for th
e three
species, resp
ectively.
Suw
ann
ayod
et al.,
2019
Cu
rcum
a lo
nga
(Tu
meric)
Simu
lium
spp
. (B
lackflies)
IV
SB
10% (w
/w) EO
form
ulatio
n p
rovid
ed 1
00%
pro
tection
for 9
ho
urs.
Tawatsin
et al.,
2006
Co
chliom
yia m
acellaria
(Seco
nd
ary screw
-wo
rm)
FPB
LD
50 against larvae w
as 0.84 μL/cm
2 48 ho
urs p
ost exp
osu
re.
Ch
aaban
et al.,
2019b
Lucilia
cup
rina
(A
ustralian
shee
p
blo
wfly)
FPB
LD
50 against larvae w
as 1.34 μL/cm
2 6 ho
urs p
ost exp
osu
re.
Ch
aaban
et al.,
2019a
Lucilia
cup
rina
(A
ustralian
shee
p
blo
wfly)
Ch
rysom
ya m
egacep
ha
la
(Orien
tal latrine fly)
Ch
rysom
ya ru
fifacies (H
airy maggo
t blo
wfly)
TA
LD50 again
st adu
lts was 94.52, 129.73 an
d 5
9.83 μ
g/fly for
the th
ree species, resp
ectively.
Suw
ann
ayod
et al.,
2019